Science topic

Macrophage - Science topic

The relatively long-lived phagocytic cell of mammalian tissues that are derived from blood MONOCYTES. Main types are PERITONEAL MACROPHAGES; ALVEOLAR MACROPHAGES; HISTIOCYTES; KUPFFER CELLS of the liver; and OSTEOCLASTS. They may further differentiate within chronic inflammatory lesions to EPITHELIOID CELLS or may fuse to form FOREIGN BODY GIANT CELLS or LANGHANS GIANT CELLS. (from The Dictionary of Cell Biology, Lackie and Dow, 3rd ed.)
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I have recently tried to do in vitro monocyte differentiation using human HSC. After addition of M-CSF, how long the cells will be matured? I also noticed a lot of cell death, are these dead cells non-monocyte cells or just due to apoptosis caused by high concentration of M-CSF? When the cells begin to attach? when the macrophage will occur?
Looking forward for your answers.
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Could you suggest some articles?
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I need to facs sort macrophages, there are many surfaces markers which are used for this purpose such as: CD14, CD68,CD64, CD11c, CD204,CD206.
I need to know why certain surface markers are preferred over the others?
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Hello Shali Ali
Macrophages can be categorized into two main types: M1 and M2 macrophages. The M1 type, referred to as classically-activated macrophages, are activated by pathogen invasion and play a large role in the immune response to foreign pathogens such as bacteria.
The M2 type, referred to as alternatively-activated macrophages, play a role in wound healing and tissue repair, and have an anti-inflammatory role.
M2 macrophages can be further divided into M2a, M2b, M2c, and M2d subcategories. These macrophages differ in their cell surface markers, secreted cytokines, and biological functions.
So, depending upon the type of macrophage you can select the markers.
For instance,
CD68 and CD11b are total markers of macrophages.
M1 and M2 macrophages have specific markers.
M1 Macrophage Marker
For M1 you may choose CD80, CD86, CD64, CD16 and CD32 as markers. In addition, the expression of nitric oxide synthase (iNOS) in M1 can also serve as phenotypic marker.
M2 Macrophage Marker
CD163 and CD206 are major markers for the identification of M2 macrophages. Related surface markers for M2-type cells also contain CD68. Compared with marker CD68, CD163 is more selective to macrophages, so CD163 can be used as a highly specific marker for M2-type macrophages.
M2 macrophages are sub grouped into M2a, M2b, M2c, and M2d.
M2a macrophages
M2a are activated by IL-4 or IL-13. M2a macrophages lead to the increased expression of IL-10, TGF-β, CCL17, CCL18, and CCL22. These macrophages enhance the endocytic activity, promote cell growth and tissue repairing.
M2b macrophages
M2b are activated by immune complex, Toll-like receptor (TLR) ligands and IL-1 receptor ligands, and they can express IL-6, IL-10, TNF-α, and IL-1β. Based on the expression profiles of cytokines and chemokines, M2b macrophages regulate the breadth and depth of immune responses and inflammatory reactions.
M2c macrophages
M2c also known as inactivated macrophages, are induced by glucocorticoids, IL-10 and TGF-β. These cells secrete IL-10, TGF-β, CCL16, and CCL18 and play crucial roles in the phagocytosis of apoptotic cells process.
M2d macrophages
M2d can be induced by the TLR antagonists, and M2d macrophages express IL-10, IL-12, TGF-beta and TNF-α.
Best.
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I need to identify natural killer cells from human peripheral blood for subsequent analysis by flow cytometry.
If someone could give me some guidance, I would be very grateful.
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I am not s superexpert, but, to my knowledge, macrophages are differentiated monocytes in tissue, not in peripheral blood, although my knowledge is not deep enpugh to exclude that this may happen in some instances. One of the most widely markers used to identify tissue macrophages is CD68. CD45 is a membrane phsophatase , present in the majority of white blood cells, not only macrophages. CD45 has been used by immunologists or pathologists to broadly identify or sorting white blood cells ( leukocytes of any type).
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I want to inject nude mice with THP1 macrophages. has anyone tried injecting differentiated THP1 into mice? Do you let them differentiate in plates? and if so how do you detach them with ought killing?
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Hello Dr. Mauricio, is there any effect of lidocaine while detaching THP1 macrophage from culture plate?
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Hi,
I am thinking about using the Raw 264.7 cell line and I was wondering about which safety measures should I use for this cell line. ATCC recommends a biosafety lab level 2, but it doesnt give much information. Other sites say that is Biosafety level 1. It seems to be a widely used cell line and I haven't seen any biosafety measures specification in publications.
I just found this publication about this concern:
Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line - PubMed (nih.gov).
Do you culture this cell line in P2 labs?
Thanks in advance.
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we use in BSL2 lab. You should probably contact safety office at your institution to find out what they classify them as.
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Hello!
We would like to treat BMDM with the caspase-1 inhibitor Z-WEHD-FMK before bacterial infection. I only found two publications suggesting either 20 or 100 µM for primary macrophages. Of course, titrating the concetration is one option. However, I will be very grateful if someone would share their experience and would recommend a concetration that worked for them. Thank you!
Best regards, Katrin
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Maybe someone is interested in this question in the future, so I will share our experiences. We did a titration of 20, 50, and 100 µM Z-WEHD-FMK starting treatment 1 h prior infection of the BMDM. We see a dose dependent reduction in the IL-1beta meaured by ELISA 1 day post Salmonella infection. We will use 50 µM for our experiments.
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Hello,
Could someone recommend me an antibody to label macrophages and M1 macrophages for an immunofluorescence on frozen heart sections ?
Thank you in advance
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Thank you Giorgio Perino and Filip Konecny for your answers!
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Hi all,
I am new to immunology field, need some help to distinguish CD3, CD4, CD8 and CD25. What are differences between them for T cells? How to decide which CDs are specific for T cells, B cells, neutrophils and macrophage.
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I commonly use CD3 to gate T cells; then CD4 and CD8 to gate T helper cells and T cytotoxic cells, respectively; Then using a CD3+CD4+ gating (Th cells) you could use CD25 and CD127 in combination to distinguish between effector T cells (CD127+CD25low) and regulatory T cells (CD127low CD25high).
You can check one of my articles for that: doi: 10.1038/s41598-019-43622-8
Best
Juanjo
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Hello, we are starting to use human macrophages, derived from MACS-sorted CD14+PBMC, for our assays. Using M-CSF we noticed, that they are proliferating at a decent rate, what made me think about cultivating them for a longer time and possibly splitting the cultures. Not having read this in papers so far, I wonder if anyone did this and if yes, what your experiences were regarding cell phenotype and senescence.
Thank you for your time,
Max
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thank you very much for your reply. What you say is exactly the issue we are struggeling with. Apart from my basic question whether there is a possibility of long-time cultivation of primary patient derived macrophages in any sort, we are currently trying out different detatchment protocols.... neither of which worked satisfyingly.
Tanking procedural cell death into account, trypsination works best, but the cells are slightly activated afterwards and some surface molecules are shred off... EDTA+cold or scraping both cause a lot of cell death. And accutase etc didn't bring any advantage at our hands, either....
Anyhow, thank you and I'll pass the greetings over at the next lab meeting,
kind regards,
Max
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Dear all,
I want to easily isolate human skin macrophages, do you think that magnetic isolation using CD14+ beads should work (after enzymatic dermis digestion) and will give only macrophages ?
Thanks
Vincent
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Yes very possible. This article at will surely be of help.
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I am differentiating THP1 cells using PMA. Which are the good markers for determining human macrophage differentiation using qPCR?
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This publication by Murray et al. (Immunity. 2014 Jul 17;41(1):14-20.) may also be useful to you:
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Hello everyone,
I am trying to freeze primary human alveolar macrophages, which are being isolated out of human lung biopsies. Currently, I resuspend 2x10^6 cells in 1 mL cryomedium, place them in a cryo box (CellCamper), and store them at -80 °C. I have tried the following cryo media:
a) PromoCell Cryo-SFM (protein- & xeno-free; based on methylcellulose, DMSO, and other cryoprotectants)
b) Sigma CryoSOfree™ DMSO-free Cryopreservation Medium (animal protein-free)
c) Culture medium w/ 10 % DMSO
d) 90 % FCS + 10 % DMSO --> often employed for BMDMs
Unfortunately, nothing has worked so far, and multiple methods (LDH; L/D staining, etc.) have confirmed I am dealing with a vast viability decrease in the first 24 h after thawing.
Does anyone have any experience with the cryopreservation of primary tissue macrophages (not MDMs or BMDMs)??? Please let me know!!!
Thank's in advance :)
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Have you also tried to store them in liquid nitrogen? Storage at – 80°C is not sufficient for long-term preservation of primary cells and causes irreversible cell damage. I highly recommend to store them in liquid nitrogen (< -150°C).
That being said, PromoCell uses their Freezing Medium Cryo-SFM to cryopreserve their assay-ready M1 and M2 macrophages. They are differentiated from fresh PBMCs, but in principle the freezing medium works for macrophages.
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I have stained spleen from mice with CD45, CD11b, CX3CR1, F4/80, Ly6G and Ly6C antibodies.
I am unsure how to use these markers to distinguish between monocytes and macrophages. It seems like the two cell types express much of the same markers.
Any advice would be much appreciated
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This is good reading starting point: 10.1371/journal.pone.0007475
Alternatively, it would have been great to have CD68 in the panel. Sometimes, you can get an impression by closely monitoring your CD14/CD16 gate
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I found 4 cell line (MD, 90196B, EL1, KMA) for that. I am looking for the differences/comparison of these cell lines.
Also, under cell morphology, all these 4 cell line show both 'monocyte'/macrophage'. Do they mean they have both type of cells under that specific catalogue ?
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Amos Bairoch thank you for the suggestions. It was really helpful. I was looking for human macrophage from spleen tissue and without any disease. Could you guide me more on this?
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Hello everyone,
I am trying to isolate bovine monocytes-derived macrophages from whole blood but I have several issues with the protocol:
1) After doing the centrifugation with Histopaque 1.077, I sometimes obtained clump of mononuclear cells at the interface between the gradient and the plasma instead of having a homogenous ring of cells. Is it normal?
2) I am using the plastic adhesion to seperate the lymphocytes from the monocytes by incubating my cells for 2 hours at 37 celcius degree in T75 flask. However, I am not able to detach all of the monocytes by using either a cell scraper or 25% trypsin+0,2mM EDTA+ HBSS. Can you help me with that?
Thank you very much
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If isolating mono-nuclear cells and then differentiating them helps, here is a protocol.
Peripheral blood mononuclear cells isolation
Whole blood was obtained by venipuncture of the vena jugularis externa into EDTA (Ethylenediamine tetra-acetic acid) coated vacutainer tubes (Becton Dickinson, Heidelberg, Germany). Peripheral blood mononuclear cells (PBMCs) were isolated from blood by using OptiPrep (60% w/v iodixanol) (STEMCELL technologies, Vancouver, Canada), as per manufacturer’s instructions with minor modifications. A range of density barriers (1.070gm/ml - 1.084 gm/ml) were tested to get maximum yield of monocytes with lowest amount of lymphocyte contamination in the pool of isolated PBMCs. For isolation of monocytes, 1.076 gm/ml density barrier was chosen from the range of 1.070 gm/ml, 1.072 gm/ml, 1.074 gm/ml, 1.076 gm/ml, 1.078 gm/ml, 1.080 gm/ml, 1.082 gm/ml and 1.084 gm/ml density barriers, after optimization. Density barriers (Annexure I) were made as per manufacturer’s instructions, by mixing OptiPrep solution with Complete RPMI1640 medium (Annexure I). Then, 54% (w/v) iodixanol solution was prepared by mixing 5.4 volumes of OptiPrep with 0.6 volumes of 8.5% NaCl solution. One part of 54% (w/v) iodixanol solution was used to adjust the density of whole blood by mixing with 5 parts of whole blood by several gentle inversions. In a 15 ml centrifuge tube, 6 ml of chosen density barrier (1.076 gm/ml) was layered above 4 ml of whole blood and 500 µl of complete RPMI 1640 medium was layered on the top of density barrier. Centrifugation was done carefully without mixing at 700 x g in a swinging bucket rotor for 30 min at 4ºC without brakes during deceleration. Cells floating at the top of density barrier were collected and diluted in 2 parts of complete RPMI1640 medium (RT) immediately. Cells were centrifuged at 1000 rpm at room temperature for 7 min and the obtained pellet was again resuspended in the complete RPMI 1640 medium. The viability of obtained cells was determined by trypan blue exclusion method. Cell suspension was diluted and mixed with trypan blue in 1:1 ratio and 10 µl of suspension was used for counting in Neubauer's chamber. Monocytes were characterized by nuclear stain Hoechst 33342 by observing their ellipsoidal (kidney-bean) shape nucleus. Briefly, 100 µl of cells were mixed with 20 µl of Hoechst 33342 stain for nuclear staining and kept in dark for 15 min. Cells were centrifuged for 2 min at 300 x g and the staining solution was aspirated completely. Further, cells were washed twice with PBS followed by observation of cells at 1000 x magnification under IX-73 Olympus fluorescence microscope.
Note: Solutions should be freshly prepared, mixed thoroughly and maintained at 4ºC before density gradient centrifugation.
Monocyte cell culture and differentiation protocol
Isolated monocytes were seeded at density of 105 cells in 12 well plates, where Poly-D-lysine (PDL)coated 18 mm round coverslips were already placed. For differentiation into macrophages of M2 (anti-inflammatory) phenotype, 500 µl of complete RPMI 1640 was supplemented with 100 ng/ml of M-CSF (Macrophage- colony stimulating factor). Cells were incubated in M-CSF for 24 hrs at 37ºC in 5% CO2, to let adhere monocytes on the PDL coated coverslips in 12 well culture plates. After 24 hrs of incubation non-adhered cells were removed by removing the media from wells and adhered cells were washed twice with 1X PBS to remove any remaining non-adherent cells. Further, cells were incubated for 7 days at 37ºC in 5% CO2 for complete maturation of macrophages. Medium was replaced after every 48 hrs of incubation with washing of cells twice in 1X PBS. Macrophages were assessed for the development of characteristic structure on 1, 3, 5 and 7 days via fluorescence staining of CD163.
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I am trying to adapt the protocol found below, but they use THP1 cells, I am using murine BMDM. I am wondering if this is possible? I cannot find purified mouse HDL or apolipoprotein anywhere and we would like to avoid purifying it ourselves if possible.
Cholesterol Efflux protocol I am trying to adapt:
To assess NBD-cholesterol efflux, macrophages were incubated in phenol red-free RPMI 1640 medium containing 5 µmol/l NBD-cholesterol for 4 h at 37°C. Following incubation, the cells were washed with PBS three times and were then incubated with HDL or apoA-1, as lipid acceptors. To determine the correlation, various concentrations of HDL (5, 20, 50 and 100 µg/ml) and apoA-1 (10, 20, 50 and 100 µg/ml) were used for the [3H]-cholesterol and NBD-cholesterol efflux assays. HDL concentrations ranged between 5 and 100 µg/ml, and apoA-1 ranged between 10 and 100 µg/ml. Subsequently, the cells were harvested after 4 h, and the medium and cell lysate were collected for the detection of FI. Triton X-100 (0.1%) was used to lyse the cells in a 12-well plate, and the cells lysate was homogenized by pipetting up and down several times. A total volume of 600 µl was then aliquoted into three wells of a 96-well plate for the measurement of FI. The percentage of NBD-cholesterol efflux was calculated by dividing the FI in the medium by the sum of the FI in the medium and cell lysate. All data were from three independent experiments, each performed in triplicate.
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Ikemefuna Ikesee limited in what way?
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I have stimulated thp-1 cells and applied inhibitor but the inhibitor did not work and I am wondering why would this coul happened, could it be because of the FBS ?
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Yes, probably you may be right. Inhibitory components of FBS could be responsible.10% FBS present in the medium contains enough TGF-β to inhibit macrophage activation. Thus, culturing macrophages in standard FBS-containing medium may be misleading because TGF-β1 inhibits macrophage activation. For more details you may refer to the research article attached below.
Best.
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I want to stain rat macrophages with GATA-6 for flow cytometry. I would appreciate if you recommend me anything?
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Sudeep Kumar Maurya thanks a lot for your support!
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Hi,
Im trying to isolate intratestinal macrophages from mouse lungs, and grow them in culture for a few days, to collect secretome. Does somebody have a good protocol for it?
I tried face sorting but the cells didn't grow well and died in the following days.
I also tried to separate f4/80 cells with macs columns but didn't get any separation.
Can someone have any suggestions?
Thank you,
Shani
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Well in that case, you should try supplementing with r-MCSF or L929 supernatant in the cultures. Macrophages are kinda dependent on this growth factor. For more information, please see:
Good luck!
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Dear all,
during flow cytometry analysis of primary monocyte-derived macrophages I observe two populations by size (see Image). Is this the usual case when differentiating primary cells in vitro using M-CSF? Most publications don´t show their first size gates and I haven´t observed this phenotype working with differentiated THP-1 cells.
Thanks!
Bianca
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Dear Alexander,
I haven´t noticed any differences between the two population looking at CD14, CD16, HLA-DR, CD68 or any polarization marker (CD206, CD40, CD163).
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Recently, we have done the macrophage antigen lpresenting experiment by co-culture of macrophages pre-loaded with OVA and CD4+/CD8+ T cells isolated from OT-II/OT-I mouse spleen. Using CFSE to stain the T cells, after 3-5 day co-culture, there is no significant differentiation of the target T cell.
Is there any probelm in the experiment? and is thre any trick in processing the sample?
Thank you very much.
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I would like to know what would be the ideal time to extract nuclear proteins from RAW 264.7 macrophages after they are treated with LPS.
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That depends on the question you want to answer. I'd expect that transcription factors will begin to be translocated rather quickly (within seconds or minutes), but only a time course will tell you what's actually happening. And when and, in which order.
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Dear all,
I already try several months to polarize in vitro differentiated THP-1 cells to M1 and M2 macrophages. I am successful in M0 differentiation and also in polarization. But sometimes cells deattach during the polarization process (48h). Here is the protocol, which I am using:
1. Seeding THP-1 cells (1,3x106 / well) in 6-well plates (2ml RPMI-medium with 3% FCS) and adding 10ng/ml PMA
3. Incubation for 72h.
4. Rest: Washing cells with 1xPBS and adding 2ml RPMI (+3% FCS) without PMA
5. Incubation for 24 hours
6. Polarization in 2ml RPMI medium (+3% FCS):
M1: 1µg/ml E.coli LPS + 10ng/ml IFN-g
M2: 20ng/ml IL-4 and IL-13
7. Incubation for 48 hours
8. Analysis: CD14, CD36, CD11b, CD80, CD206 and CD209
During the second day of polarization cells deattach again (in all wells M0, M1 and M2). And I do not know why. I have already tested lots of conditions:
a. different cell numbers
b. different cell passages
c. new ordered versus "old" PMA
d. I also tested different PMA concentration, different incubation times and resting times for in vitro differentaition (analysing cells after 24h rest, without polarization phase). And I do not want to increase PMA due to its potential to shift THP-1 to M1 macrophages.
I started these experiments with medium containing 10% FCS. Reducing FCS to 3% during the whole experiments is the only conditions which showed significant better results. Using only 3% FCS caused very strong attached macrophages with their typical morphology (not seen when using 10% FCS). However, it worked three times and now it does not work (even with freshly thawed cells).
Could it be a medium issue? For thawing we are using Gibco RPMI with 20% FCS until cells are in exponential growth phase. Afterwards I change the medium to Sigma with 10% FCS (both without antibiotics). When the 3% FCS during experiments caused the significant better results, cells were changed at the day of seeding from Gibco to SIgma medium.
I do not know what else I can check. Has anyone some suggestions??
Thank you very much.
Christian
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1) The density of cells per well
Totally agree with Anatoliy on the density. I had the same issue at the beginning of my experiments when some of my THP-1 cells detached. In my experiements, I was working on M0 and M1 phases. I ran some optimizations on the cell density but on 96-wells and 24-wells plates. Here's my parameters (perhaps you can adapt them to your 6-well plates):
RPMI-1640, 10% FBS, 1% L-Glutamine
96-wells - 4 x10^4 cells/ well in 100 μL
24-wells - 2.5 x10^5 cells/ well in 500 μL
PMA - 200 nM
PBS - use warm PBS to wash the cells gently
Incubation time: 48 hours
Use fresh PMA (prepared in small aliquots, do not re-thaw)
2) PMA stage to PMA(r) stage, with less % of FBS
One major change that I eventually did was prolonging the incubation time without PMA for 3 days, to help with the attachment. Check this paper:
RPMI-1640, 1% FBS, 1% L-Glutamine
LPS - 1 μg/mL of E. coli 0111: B4
Incubation time: 72 hours (as according to the paper)
3) The optimal condition of THP-1 cells
Culturing THP-1 cells were quite tricky sometimes and similarly, I ran numerous times of optimisations and I cannot conclude consistent parameters for months. The other tip that I can share is I did not use or culture the THP-1 cells more than few cycles. What I eventually did was buying fresh THP-1 cells from ATCC and prepared many fresh stocks. From that point, I eventually was able to produce better and more consistent outcomes.
Hope it helps! I understand the feelings of optimising and working on the THP-1 cells for months but with so many inconsistencies!
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Cell differentiation was induced via a 6-h exposure to 185 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma–Aldrich) in DMSO. Cells were then polarized toward M1 or M2 phenotype by incubation for 48 more hours with INF-γ (20 ng/ml, Immunotools) and LPS (100 ng/ml, Sigma–Aldrich) or with IL-4 (20 ng/ml, Immunotools) and IL-13 (20 ng/ml, Immunotools), respectively (Tjiu et al., 2009; Maeß et al., 2014), still in the presence of PMA. Cells used for the resting condition were kept in the presence of PMA for 48 more hours in normal growth medium. CM were then prepared by keeping polarized as well as resting cells in serum-free RPMI without stimuli and no PMA for 72 more hours.
Basically, what I'm seeing are wells with M1 macrophages that dwindle in number over the 72 hours post-exposure to INF-γ and LPS. I'm not sure if this matters, but their distribution is typically only at the periphery of the well by the end of that time (images attached are of this area). Also, for some reason, their association (phagocytosis) with beads is lower than both M0 and M2. What could I be doing wrong?
Troubleshooting steps:
-Order fresh reagents & prepare new aliquots
-Tried another vendor
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Dear Cody,
I observed the same issue with primary human monocyte-derived macrophages. For this primary macrophages, the cause of death was a prolong exposure to the polarizing cytokines namely LPS and IFNy (M2 polarization with IL-4 survive better). Hence, we moved away from exposing macrophages to the polarizing agents for more than 48h. The polarization M1 markers that I use to identify M1 macrophages remain highly expressed for several days without LPS/IFNg stimuli but the expression levels decreased over time. I perform my assay 7 days after polarization and I can still distinguish M1 from M2 macrophages, but M1 macrophages on day 1 without IFNg/LPS have higher expression of polarization markers compared to M1 macrophages on day 7. Survival signal M-CSF remains in the culture - without it, macrophages are dying.
Good luck with your experiments!
Best,
Bianca
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Have anyone detected the CAR expression on macrophage cells?
Which method is preferred to use?
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Follow this link, you can take some information from this paper regarding CAR.
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I'am culturing bone marrow derived monocyte with 20ng/ml M-csf. The in vitro monocyte with M-csf will mature into macrophage. I'm wondering that is their a way to maintain the monocyte in vitro without maturing into macrophage and just let the monocyte poliferate.
Is it work if I just use basic cell culture medium withou M-csf to let the monocyte survive and poliferate?
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I agree with Gaurang Telang .Monocytes cultured for 4-5 days in the presence of M-CSF are considered macrophages.
You could try to control some of the factors during culture of monocytes. Maybe those would be helpful.
First, strict pH control is required, particularly during the first 24 hr of culture. Variations from a range of 7.3 to 7.5 may seriously impair monocyte viability, especially when more alkaline. Therefore, it is recommended that the monocyte medium be preequilibrated in the CO2 incubator prior to use.
Second, complete humidification of the incubator is mandatory, as monocytes are very sensitive to slight evaporation of the media.
Third, the presence of contaminating lymphocytes or platelets has an unpredictable effect on the monocytes. Therefore, consistency of success depends on use of a purified cell population from the beginning of culture.
Under these controlled culture conditions monocytes could probably remain in culture. Cultures are fed every third or fourth day by replacement of half of the medium with fresh complete medium. Monocyte cultures will start to show deterioration during which time the cells will detach from the substratum and begin to die despite continued feeding. Monocytes will not divide at any time during routine culture.
For more information on culturing human monocytes you may refer to the link below.
Chapter 4 OBTAINING AND CULTURING HUMAN MONOCYTES
By Steven D. Douglas, Steven H. Zuckerman and Samuel K. Ackerman
Pages 33-42.
Best Wishes.
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I would like to transfect THP-1 derived macrophages with ds- and ssRNA and -DNA and would like to use a transfection reagent that does not require electroporation since we don't have an electroporator. Any suggestions welcome. Thanks.
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Yeah, I was thinking about this. Will try both approaches.
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I've been trying to cultivate BMDM into macrophages since a weak, and I'm using DMEM with 10% FBS and 5% pen strep. I changed the media three or four times and rinsed it with 1X PBS once. Although it is the eighth day of this culture, I have yet to notice a difference in the confluence. I'm using m-CSF to differentiate macrophages.
please suggest something that works for me!
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Bone marrow derived monocytes (progenitors) are what you isolate from the marrow, they won't proliferate any further, all you can do is force them to a macrophage lineage using MCSF then either pro or anti-inflammatory stimuli. If anything after a few days you will see slight loss in cell number however after about 7 days you should notice that the cells become almost "stick or rod" like and will begin to cluster and form colonies, these are macrophages and from there you can force them to either pro or anti-inflammatory depending on the stimulus you give them. For pro-inflammatory LPS/TNFa cocktail for 24 hours works great, for anti-inflammatory an IL-4/IL-10 cocktail for 24 hours.
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later in down stream i have to use these proteins for pulldown. so kindly address me how to extract them in native form ? thank you
regards
ravi
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A common method is to dissolve the membranes containing the proteins with a non-denaturing detergent, such as dodecylmaltoside, octylglucoside, and some others.
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Dysregulation of pro-inflammatory cytokines promotes immune-mediated injuries 1.
Both epithelial-cell proliferation and an increase in macrophages in the lung are associated with SARS 2. Pathogen-associated molecular patterns (PAMPs) such as 2019 novel coronavirus, proinflammatory cytokines, and lipopolysaccharide (LPS), cause macrophage transition. Macrophages activated in this transition termed M1 macrophages that promote inflammation 3. PAMPs are primarily sensed by members of the Toll-like receptor (TLR) family and this sensation activate transcriptional factor NF-κB that promotes secretion of proinflammatory cytokines 3. Activated or infected immune cells secrete excessive proinflammatory cytokines including MIP-1α, RANTES, IP-10, IL-1-6-8, TNF-α, TGF-β1, MCP-1and MCP-1 4. These cytokines promote severe lung injury 1.
Thalidomide is an immunomodulatory agent with strong antiangiogenic properties with COX-2 inhibitor celecoxib. Thalidomide inhibits the mRNA encoding such as TNF-α and VEGF. In addition, thalidomide modulates activated or irregulated NF-κB, resulting in suppressing severe lung injuries.
However, we are ordered not to conduct clinical trials with thalidomide and celecoxib
by Ministry of Health, Labor and Welfare, Japan. Because thalidomide is the dangerous drug.
I think it would be difficult to save the patients with severe pneumonia who could be saved with this regimen in the future. I would like you to recommend to the Japanese Ministry of Health, Labor and Welfare to take appropriate measures in Japan from the Form.
Please post your opinion in the form that Japanese people could have effective treatment for 2019-nCoV infection. https://www.kantei.go.jp/jp/forms/goiken_ssl.html
1. Jiang Gu , Christine Korteweg
Pathology and Pathogenesis of Severe Acute Respiratory Syndrome
Am J Pathol, 170 (4), 1136-47 Apr 2007
2. John M Nicholls , Leo L M Poon, Kam C Lee, et al
Lung Pathology of Fatal Severe Acute Respiratory Syndrome
Lancet, 361 (9371), 1773-8 2003 May 24
3. Yannic Nonnenmacher, Karsten Hiller
Biochemistry of Proinflammatory Macrophage Activation
Cell Mol Life Sci, 75 (12), 2093-2109 Jun 2018
4. Chaolin Huang, Yeming Wang,Prof Xingwang Li, et al
Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China
The Lancet Journal
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I am looking for a macrophage human cell line. Preferably one that is already adherent vs a cell line that requires differentiation.
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You can use RAW 264.7, THP-1, U937
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I have been trying to evaluate the role of Kupffer cells (KCs) in my in vivo model and therefore I want to find a way to deplete this cell type. I have been looking in the literature and found some different treatments previously used such as gadolinium chloride and clodronate liposomes.
My current problem is that I dont want to deplete macrophages from other organs, specially macrophages from the spleen. Therefore I wanted to know how specific are those treatments to KCs and/or how to make sure that the splenic macrophages are functional.
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Your best option will be to use an Clec4f–DTR mice. Then you can specifically deplete KCs.
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Hi guys,
I am currently writing my dissertation on the phagemid based delivery of CRISPR.
I'm currently looking at the limitations, specifically now looking at the immune response against phage. However, I'm struggling to see how phage is actually administered and where the immune response would occur.
Is the main route of transmission through the bloodstream, where antibodies and macrophages act on the phage and form a response or does this not occur?
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Hello Ben,
Intravenous administration of phage therapy started in the 1940s.
Kindly read article titled " Safety and efficacy of phage therapy via the intravenous route" DOI: 10.1093/femsle/fnv242.
Furthermore, topical application also demonstrated no side effects, see the attached article
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I am looking for a method to assess the function of cancer cells and macrophage co-culture. rather than inducing the macrophage to either only M1 or M2, I would like to see what is the effect of coculture between a spectrum of macrophage and the cancer cell in vitro. I think this can recapitulate more the in vivo microenvironment where both M1 and M2 macrophages co-exist.
I am using both the J774 and THP-1 cell lines. May I know if there is some well-established protocol/ published article that tried inducing the differentiation of both M1 and M2 at the same time?
Thanks!
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I think CCL7 is one such chemokine that can induce both M1 and M2 in-vitro. I am not sure there might be more. Check this article - Macrophage Polarization: Different Gene Signatures in M1(LPS+) vs. Classically and M2(LPS–) vs. Alternatively Activated Macrophages
But if this has not been shown already, perhaps you would first like to validate protocol and then verify the mixed phenotype obtained.
I agree that its more valuable to study the spectrum in case of macrophages. Did you tried inducing them separately and later mixing for experiments ? You could also sort the mixed population to work with.
Good luck!
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Hello,
I am looking to design a proteomics experiment looking at three treatment concentrations (Control, low-dose, high-dose) and two timepoints (24h, 48h) in an attempt to discover an unknown mechanism for lipid accumulation in THP-1 macrophages. I have never stepped into the omics world before so I thought I would start by asking:
What do you know now that you wish you had known when you started?
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Hi Braeden Giles
Mass spectrometry is a very sensitive and accurate method to determine the precise molecular weight of a protein.
Because it is very sensitive and is amenable to rapid processing of many samples, it is becoming very popular for determining the different proteins present in complex samples (e.g., the proteins in a given cellular organelle, separated by two-dimensional gel electrophoresis), in what is known as a proteomics analysis.
Techniques have now been developed by which proteins separated in two- dimensional gels can be digested within the gels using endoproteases and then injected directly into a mass spectrometer for analysis of the resulting fragments.
Best
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Hi I am interested in IF staining macrophages in frozen colon sections.
I chose F4/80 as it is a pan-macrophage marker. However, I have hard time staining macrophages in mouse colons.
Typically, I fix freshly excised colons in 4% PFA for 2h at RT followed by sucrose priming (30%) overnight and flash-freezing samples. I think this protocol is pretty standard and it has worked well for staining other antigens.
I have used two biotin-conjugated antibodies for staining F4/80: 1)Thermofisher, 13-4801-82 and 2) BD Biosciences, 565635. Both of them resulted in very weak or almost no staining even at low dilution rate that Datasheet suggests.
If anybody who works a lot with macrophages and routinely stain macrophages on frozen sections, could you please share the protocol?
By the way, I cannot use organic solvent-based fixation (e.g. methanol/acetone) as other antigen I co-stain with F4/80 are sensitive to that. So I can only use aqueous fixatives.
Thanks,
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Thanks Ute,
I did not think of changing fixation time as this condition has worked for other antigens. I will try fixing longer and see how it goes before touching on other staining techniques. Thanks for your suggestion!
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Hello,
Can anyone please suggest the markers and protocol for FACS staining for macrophage from PBMC in human.
Thank you.
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Thank you very much everyone for you suggestion.
Martin Waterfall & Lora Benoit - Firstly, thank you very much for your suggestion.
I would like to stain pan macrophages(M1+M2), M1 and M2 from PBMC's.
Martin Waterfall & Lora Benoit - I would like know you comments on following panel for staining pan-macrophages, M1 & M2
CD45+CD3-CD19-CD14+CD16- (Monocytes)
CD45+CD3-CD19-CD14+CD16+ (Macrophages)
M1
CD45+CD3-CD19-CD14+CD16+ CD80+
CD45+CD3-CD19-CD14+CD16+ CD86+
M2
CD45+CD3-CD19-CD14+CD16+ CD163+
CD45+CD3-CD19-CD14+CD16+ CD206+
Thank you very much for your recommendations and comments.
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I am trying to culture macrophages from the bone marrow of mice. I always worked with macrophages and had no problem culturing them but recently it has been hard to get them as most of them do not attach to the plate and die after one day (They look small). After isolating bone marrow cells and incubating them for 1 day, I collect non-adherent cells and incubate them for two more days to get macrophages. Here is the image of my cells (40 x) that I am expecting to see macrophages. (The black dots are the background of the microscope).
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okay, fresh BM and 50ng/ml of M-CSF sounds good, even better than L929 S.N.. Can you verify that the M-CSF itself is still intact/working as intended? Did anything else change recently? (Culture plates/flasks, media?). Do you perform a lysis step of red-blood-cells in the isolation procedure of the bone-marrow cells?
Regarding the purity: I don't want to tempt you to overthrow your protocol if it is already established in your lab and worked nicely in the previous attempts. However, pure monocyte/macrophage cultures can be easily achieved by making use of their adherent characteristics. Whenever you start another bonemarrow macrophage differentiation, maybe try the following in parallel:
  1. Seed approx. 1 x 10^7 BMs in 10ml of your differentiation media into a T75 flask (or equivalent density in another flask; ideally some that are specially designed for adherent cells)
  2. Two days after seeding, simply add 10ml fresh differentiation medium on top
  3. Four days after seeding, take of 10ml of the medium (without disturbing the adherent cells) and just add 10ml fresh differentiation medium on top
  4. Six to seven days after seeding the cells are ready. Take off all of the media (containing non-adherent cells), rinse the adherent cells once with PBS, detach the adherent cells and continue your assay. At this stage, the adherent cells should have a high purity of macrophages (we check them via flow cytometry using CD11b and F4/80 as differentiation markers and usually get 90-95% purity).
Sorry that I don't have an exact answer to your question yet but maybe this helps a bit :)
Best regards
Felix
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I have RNAseq data from some human WT and KO macrophages. I have a list of DEGs from this comparison.
However in addition I have been asked to demonstrate using the data I have that they (both WT and KO) are more like macrophages than other PBMC and then also secondly to compare them to other macrophage subsets.
I have searched the GEO databases and there are datasets available that compare many human PBMC and I am sure I can also find some comparing macrophage subsets however how do I practically then compare may data with these datasets to demonstrate this?
Any help very much appreciated
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Hatty Douthwaite To isolate and sequence RNA inside the nucleus, snRNA-seq employs a rapid and gentle nuclear dissociation technique. The dissociated cells are then suspended and gently lysed, allowing the cell nuclei to be isolated from the cytoplasmic lysates through centrifugation.
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Can someone share some review article or link, on comparison of all the available Macrophages Cell Lines?
I need to know Pros and Cons of the Macrophage Cell lines.
Thanks in advance.
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  • THP-1 are human monocytes that must be differentiated into macrophages. They are popular and reliable for many types of assays. They grow in suspension culture before differentiation and are very sensitive to perturbation. Suitable for viral transduction and, if you are lucky, nucleoporation for gene delivery.
  • RAW 264.7 are mouse monocyte/macrophages that grow in adherent culture and are easy to work with. They are reliable for a variety of normal macrophage responses, but do not express the protein ASC and are thus incapable of appropriately activating the NLRP3 inflammasome. Use with caution. Suitable for transfection (because they cannot activate NLRP3...).
  • J774A.1 are mouse monocyte/macrophages that grow in adherent culture and are easy to work with. They are reliable for all normal macrophage responses and have a complete inflammasome pathway. They are sensitive to perturbation. Suitable for viral transduction for gene delivery.
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The aim of my study is to cultivate different types of macrophages, and to put them in co-culture with fibroblasts. I already arrived to differentiate monocytes in M1 and M2a macrophages. But my problem is that, when I put macrophages on my fibroblasts, I don't see any effect. Maybe it's because my macrophages are not activated ? What should I use to activate and to make them synthesize cytokines ?
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Two questions for you:
  1. How did you differentiate your cells?
  2. What cytokines are you interested in seeing?
E. coli LPS (I use O111:B4) at a concentration of 1-10 ng/mL will induce several cytokines to release from macrophages within a few hours. A great example is TNF. However, other cytokines will be expressed but not released, such as IL-1b. It will remain intracellular upon LPS stimulation until induced to release by an inflammasome activator.
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Having a bit of trouble with working out some calculation's.
I have purchased 1mg PMA and plan to dissolve in 1ml DMSO to get a concentration of 1mg/ml.
My plan is to aliquot thESE and freeze. Perhaps 1ul in each vial?
However i am unsure of how many mls of media to add to each aliquot to give the final concentrations of 2.5ng/ml, 5ng/ml, 10ng/ml, 20ng/ml and 50ng/ml?
Probably quite a simple calculation but any help would be greatly appreciated!
Thank YOU
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Louise Dolan you're welcome.
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Hi all!
I want to carry out immunofluorescence to detect macrophages. Is M-CSFR 1 specific to identify macrophages? I am a little bit confused because I have read that this receptor is also expressed by monocytes and apparently M2 macrophages, so I don't know if I can check specifically the whole macrophages population with this strategy.
Thank you very much for your help!
Rosa
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Hello Rosa Gallo
The major site of CSF1R expression is in macrophages. In hematopoiesis, CSF1R is upregulated during monocyte differentiation, but is downregulated during granulopoiesis. CSF1R is expressed predominantly in committed macrophage precursors, monocytes and tissue macrophages.
There are several macrophage cell surface markers which are commonly used, but the exact marker that you may have to use will depend on the condition of their local environment as macrophages exhibit phenotypic heterogeneity, and they are also able to modify their immunological response according to individual stimuli.
Best.
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Hey everyone. I have been trying to count macrophages with Fiji but it has only been manually up till now. I was wondering whether there is a specific plugin that I could use in order to make counting faster and more accurate.
Thank you!!!
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Hi Vania Nicole Byrne . If you are able to threshold your images, you can use the particle analyzer to automatically count macrophages. Check this video tutorial from my channel:
If your images are z stacks, you can do this:
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I know this method published in PLoS ONE but do not whether I will be able to use it as I do not have matlab. I will try to use ImageJ or FIJI...
Kozlowski C, Weimer RM (2012) An Automated Method to Quantify Microglia Morphology and Application to Monitor Activation State Longitudinally In Vivo. PLoS ONE 7(2): e31814. doi:10.1371/journal.pone.0031814
Thanks in advance for your help.
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Try this one.
It's easy to apply, the only thing you need is FIJI.
Regards
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I am working with bovine monocytes to assay innate immune response against to S. aureus
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Hi Marco,
It seems that PMA is not a good option to differentiate bovine monocyte to macrophages. Inasmuch as we did not worked with bovine monocyte/macrophages transition but rather with monocyte-derived macrophages in human THP-1 cell line (please find about it within http://www.xenobiovir.com/), we collected data on other species models, and found the following protocol as regarding bovine mononuclear cells differentiation into macrophages. Bovine mononuclear cells were separated by density gradient centrifugation; whole blood was centrifuged at 300 g for 15 min, Buffy coats were diluted 1:1 in warmed Dulbecco’s phosphate-buffered saline and over-layered onto Ficoll-Paque, then centrifuged at 400 g for 30 min. Contaminating red cells were removed using erythrocyte lysis buffer containing 0.15 M ammonium chloride. Cells were then separated to isolate CD14+ cells. Cells were suspended in RPMI with glutamine supplemented with 10% FCS, containing 200 U/ml penicillin and 200 mg/ml streptomycin at 5–20 x 105cells/ml (1–4 x 105cells/well) in 96-well plates in triplicate for biochemical assays or in 24-well plates for RNA extraction (3–4 × 106 cells/well). Cells were incubated at 37 °C in 5% CO2, medium being replaced every 2 days, until cells achieved 80% confluence and they had matured into monocyte-derived macrophages (MDM), at 6–12 days culture.
MDM or SM were stimulated by addition of 50–2000 ng/ml LPS from E. coli 0111:B4 and/or 20-100 ng/ml bovine IFNγ or with 10-40 ng/ml recombinant bovine IL-4 and/or 10-20 ng/ml recombinant bovine IL-13. Following stimulation for varying periods of time (12–72 h), supernatants (200 μl) were collected and stored at -20 °C for NO, chitinase and IL-6 assays. Cells in 96-well plates were lysed with 100ul 1% Triton X-100 in PBS for 20 min at room temperature and lysates stored at -20 °C prior to arginase assay. Cells in 24-well plates were used for RNA extraction.
Hope the above would help you in your studies.
All the best,
Ilya
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I have isolated cells from muscle tissue, culture them in adherent flasks and want characterize them. CD56 is a marker for myoblasts, but also for natural killer cells and macrophages. Is it expected to have contaminating population of NKC or macrophages present among myoblasts to test positive for CD56? The cells are characterized after the expansion on plastic.
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I have never cultured myocytes, but I have worked with macrophages and NK cells. Macrophages and NK cells co-express CD45 [also known as hematopoietic cell phosphatase] and C56. You can exclude them by excluding CD45+ cells from your analysis. Of the two, I would expect macrophages to be more prevalent than NK cells.
That said, I would not expect NK cells to grow under the conditions that support myocytes. NK cells need the cytokine IL-12 to grow. Macrophages are more plastic in their growth requirements, but they do need a source of either GM-CSF or M-CSF, which I also suspect are not present in myocyte culture conditions.
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I am currently growing the THP-1 cell line for a research study on inflammatory markers and the cells don't seem to be growing very well. I am now in my third week of growing them and I have not seen them at confluence yet thus they haven't been subcultured yet either. I use RPMI-1640 from ATCC supplemented with 10% FBS and pen-strep. I have been changing media about every 2 days but have recently started letting them sit 3 days to try to let them grow more without being interrupted. I have also started seeing a few cells differentiate into macrophage like morphology taking on a more skewed tubular look. I know that some of these observations are "normal" and "expected" of this cell line in the first weeks (growing slowly, differentiation due to quality of media, growing in aggregates, etc.) but I am looking for any suggestions on how I can try to improve the ways in which I am culturing this line.
(Fyi, I am using a THP-1 cell culture protocol authored by Cordula Hirsch and have linked it as well for reviewal. I have made a few changes (pen-strep) but otherwise the protocol has been followed very closely).
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Hi Wesley Quist,
When you first revive the THP-1 cell, are you using an untreated T-75 flask or an untreated T-25 flask? If you use T-75 flask, that explains why it grows slowly, cause it requires cell-to-cell interaction in order to grow and multiply well. I will recommend you to revive them in T-25 flask and let them incubate in a standing position instead of laying flat, as it increases the cell-to-cell interaction. Additional FBS helps to improve the cell viability and may help to grow better. Hope it helps you.
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Hi! I am trying to polarize RAW 264.7 macrophages on the 7th pass (ATCC) with 100 ng/mL LPS (Sigma) for M1 and 40 ng/mL IL4 (Sigma) for M2. They were plated at a density of 20,000 cells per well, allowed to stabilize for 24h and exposed to LPS and IL4 for 1h.
When analyzing by qPCR, I found no difference in gene expression of any of the following genes: TNF A; ARG1; IL1B; iNOS; HO1; NRF2 and GMCSF.
These are protocols established in the literature and were followed to the letter, but I could not prove the polarization process.
Should I change markers? Was the exposure time to LPS and IL4 short? Concentration should be higher?
Thanks!
Regards,
Antonio.
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Hi Antônio Secco Martorano , If you thinking about the makers of M1 and M2 polarization, This paper will give you a clear idea in choosing markers (Novel Markers to Delineate Murine M1 and M2 Macrophages; DOI: 10.1371/journal.pone.0145342). According to them, TNF a, Arg1, and IL-1B are not distinctive markers (Fig2). You can try some other distinctive markers such as Fpr2, Gpr18, CD38 for M1 and c-Myc, Egr2 for M1. In one of my publications, I have used them and I used M1/M2 gene expression ratios to delineate the activation state (Cytosolic β-catenin is involved in macrophage M2 activation and antiviral defense in teleosts: Delineation through molecular characterization of β-catenin homolog from redlip mullet (Planiliza haematocheila).
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I have THP-1 on an early pass (they are 5th pass). Most of them exhibit a round, single-cell morphology, as expected. However, some of them start to elongate (some kind of tuber-like shape). This happens every time, so I was wondering what could I do to avoid it?
I have also noticed that after many passes, some of the cells become way bigger than most of them, which I would like to understand as well.
I keep them under 1.000.000 cel/mL concentration and clean from any kind of contamination.
Thank you in advance for you time and help.
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Please refer to the research paper attached. The investigators demonstrated that the culture conditions of THP-1 cells can alter their response to PMA.
Highlight:
In this study we evaluated the effect of culture conditions on the heterogeneity of the THP-1 cell line. In low-density cultures, we found, as reported in initial studies, these cells are homogeneous, characterized by small cells with low density are CD14- and differentiation with PMA does not increase CD14 expression. However, cells kept for an extended period of time at high cell density, the culture becomes heterogeneous with a high number of big cells, increased density, and high percentage of CD14+ cells. This culture is highly sensitive to PMA and CD14 expression increases significantly……
They have concluded that culture conditions of THP-1 such as concentration and time is extremely important to take into consideration before starting differentiation studies using THP-1 cells.
Hope this helps.
Good Luck.
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I will be using human tissue to isolate macrophages to study its M1/M2 status. As we do not have a flow cytometry, I have to take my sample to different place to do the experiments. So, I need to store macrophages isolated from min 6 samples and then process it do the flow cytometry experiments.
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Thanks Nathan Kieswetter for the reply. I will try to run a pilot first and proceed accordingly.
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I am looking for M1 macrophage marker that is unique and be expressed only by M1 and not by M0 nor M2. the research papers show different ones like CD80, CD86, CD64, and CD40. The monocytes are from the peripheral blood of healthy donors.
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You might try CD115 (c-fms) which is the receptor for M-CSF. I know that mouse M1 macrophage express CD115.
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Clinically, How can we explain a patient's elevated CRP levels when he have a positive microbiological culture? (gram negative). Meanwhile, the WBCs and macrophages are normal or slightly below normal
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CRP can elevated due to any acute inflammation from bacterial causes or other causes. CBC may be change or not in Acute inflammation.
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I collected PBMC and cultured the cells in full medium with 10 ng/ml PMA in order to polarize monocytes to macrophages without isolating the monocytes from PBMC. After 24h I washed and discard non-attached cells. However, the background is full of small and long particles. What are these particles and do they affect polarization? If it affects polarization how can we discard them?
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DearAhmed,
This is bacterial contamination.
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While performing proteomic analysis of MEFs (four passages, P4), we have detected certain macrophage-specific integrins. Does anyone know if is common to have macrophages growing in primary MEFs cultures (passages 4-5), or if MEFS can express this type of integrins?
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Hi, you shouldn't observe macrophages in MEF. As Ellen stated, there is overlapping of integrins between cell types. Re-check with specific markers of macrophages and you may share images with us if you want. On another hand, you may ask the other researchers using the same batch of MEF.
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Of course, we are not talking about the survival of Promastigote
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I think the most reliable way to analyze the viability of Leishmania in macrophages is to use IF microscopy. There is a nice publication about such an in vitro assay for drug discovery against Leishmania donovani, which includes infection of THP-1 macrophages with GFP-expressing Leishmania following analysis by IF microscopy. I hope this will help you.
Tegazzini D, Díaz R, Aguilar F, et al.
A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani. Antimicrob Agents Chemother. 2016;60(6):3524-3532.
doi:10.1128/AAC.01781-15
Good luck!
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Hello , i have 1 question ? I study the migration of macrophages. Can the cells migrate after the THP-1 cell induced with PMA (phorbol 12 myristate-13-acetate).
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Please see attached publication for a side by side comparison of THP-1 and PBMC derived macrophages in a standard chemotaxis assay using HUVECs.
I hope that helps when designing your experimental protocol.
All the best & kind regards,
Michael
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Dear all,
i have been trying to isolate microglia of adult murine brain . i used a percoll density gradient; unfortunately it worked only in the first time for me, very well. after that.. it failed. no layers visible. no cells at the aimed position in the gradient.
#i would be very happy if you could share your protocols with me. i need a simple method in order to get macrophages of a brain.
#hope you can help.
regards
silke
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Dear Silke,
in case you still have some troubles with microglia isolation using different percale percentages, you could also try to isolate the microglia using Miltenyi Kits (Neural tissue dissociation Kit P) followed by 30% percoll and Macsing. More details you can find in our publication here:
This worked for us pretty well and is avoiding the tricky percoll layering.
All the best,
Anne
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In order to get M2 macrophages, I used THP-1 with 100 ng/ml PMA for 48h, then the cells were rested for 24h in a fresh medium without PMA. Then I put 20 ng/ml IL-4 for 48h. However, after staining the cells with CD206 for M2 and CD11b for M0, I only get about 7 % M2 and 75% M0. Some research papers and reviews recommend using PBMC monocytes instead of THP-1 because THP-1 cells are unable to be polarized to M2 and that approves the results that I get.
Have you ever used THP-1 and gotten more than 50% of M2 or TAM M2-like macrophages? Or I have to use PBMC monocytes. The problem in cell type or markers and cytokines or both? My purpose to get only more than 50% M2.
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After initial priming with 100 ng/ml PMA for 48h and then resting the cells in fresh medium for 48h, treat the cells with a cocktail of 20ng/ml IL4 and 20ng/ml IL13. Culture the cells for another 48h. Wash the cells and culture the cells back in fresh media fo another 48h before detaching them for staining and acquiring them on flow.
As these cells are highly adherent, detaching process will result in cell breakage and cell death. Using Trypsin may hamper cell surface markers. This will affect your results on flowcytometry. So, for detaching the cells, try incubating the cells in ice cold PBS w/o calcium and magnesium and 5%FBS on ice for 45min. Gentle scrapping will help you get cells in better condition.
Alternately, you can try using wester blotting, imaging. CD209, CD163 and TGM2 are good M2 markers to rely on. PCR is also an option to consider
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I am trying to set up an in vitro experiment where I co-culture peritoneal macrophages and T cells together in the presence of HMPV infection. After incubating the macrophages and T cells together, I harvest the cells and run flow cytometry looking for T cell activation via Ki67 and CD44 markers.
I have repeated this experiment three times, adjusting the conditions between the three replicates and each time I see that the uninfected macrophages and T cells are more activated than the infected groups (i.e expressing more KI67 and CD44).
I tried removing mCSF from the media after setting up the co-culture since I thought this might be activating the macrophages. I also tried various lengths of times for the co-culture ranging from 48hrs-4 days. I added IL-2 to the media to help promote T cell survival. In addition, I use a 96 well u-bottom plate and added the macrophages: T cells in a 1:10 ratio to ensure adequate T cell activation.
Please let me know if you have any suggestions for what I should try next. Thank you!
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Dear Olivia,
your protocol for peritoneal macrophage isolation sounds good to me. I am a little bit curious about the purity. Before magnetic cell sorting we only get 40% of macrophages from the peritoneum (gated to CD11b and F4/80).
The other cells are mainly B cells and a few neutrophils. So when you plate your whole lavage without seperating the peritoneal macrophages, you will get a co-culture of your T cells with a mix of cells but for sure not only with peritoneal macrophages.
B cells and Neutrophils might not only influence your macrophages, but also your T cells. If not with secreted cytokines or other molecules, just because they will die over night and release DAMPS. So basically you confront your T cells with a undefined mixture of cells and molecules released into the medium.
I think a pure culture of peritoneal macrophages will solve your problem. And then you can infect them and you will only have the PDL1 upregulation as factor that might influence your T cells.
I hope that helps you. For any more questions please feel free to ask any time :)
All the best and stay healthy,
Marc
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I am using the B16-F10 cell line as a model for melanoma in mice. after treatment, i see an increase in the ratio of M1/M2 macrophages in tumor tissue. i suspect that the treatment I use either reprograms M2 TAMs to M1 or increases M1 migration into tumors from other sites? I was thinking of using a drug (after the tumor reaches 100mm3 in size) to inhibit the recruitment of any new microphage into the tumor thus I can distinguish whether TAMs already in the tumor are reprogrammed to M1 or M1 are migrating from other sites to the tumor.
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By decreasing extracellular acidity (e.g. bicarbonate) and decreasing intracellular alkalinity (e.g. amiloride) you will substantially decrease migration.
Another useful drug is dasatinib that inhibits Src phosphorylation and limits the cytoskeleton activity involved in migration.
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Hi everyone,
does anyone know the best condition to isolate and culture monocytes and monocyte-derived macrophages? Many papers suggest DMEM, others RPMI + 10% FBS. I am culturing these cells in RMPI+10% FBS for 2 weeks already. I started by observing WBCs (presumably monocytes), which eventually differentiated in macrophages (spindle-like and/or "fried egg" phenotype). Over the second week I noticed a decrease mostly in macrophages number and cell growth seems to have slowed down. I've searched for these specificities in the literature, but couldn't find anything specific to the duration of these cells in culture.
Any help would be much appreciated,
Best regards,
Luisa
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Macrophages are derived quickly from monocytes by using Phorbol Myristate Acetate (PMA), this happens with a period of overnight, thereafter you can store and revive your macrophages though it mustn't be for more than three months. With media RPMI supplemented 10% FBS will do.
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I have tried to differentiate THP-1 to Macrophage M0 on polyacrylamide hydrogel which is coated with 100ug/ml collagen and cells become adherent but their morphology didn't change. Also, I used CD68 as Macrophage's marker and it didn't work (IF). Do you have any suggestions?
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Dear Mubarak!
Please You look at the articles:
Hydrogel fabrication
Fibrin hydrogel substrates were fabricated at 2 mg/ml by mixing soluble fibrinogen from human plasma (EMD Millipore, 341576) mixed with human thrombin (0.4 U/ml; EMD Millipore, 605190) and pipetting it gently into tissue culture wells (with or without glass coverslips) to avoid bubbles. The hydrogels were allowed to form at 37°C for at least 30 min before seeding cells. The collagen gels (2.0 mg/ml) were fabricated using rat tail type I collagen (Corning) according to the manufacturer’s protocol. Briefly, collagen was mixed with appropriate amounts of 10× phosphate-buffered saline (PBS; Lonza), 1 M NaOH, and water. Five percent PEGDA hydrogels (200 molecular weight) were made by mixing equal volume of 0.05% Irgacure and 10% PEGDA in PBS. After dispensing the mixture into the well, it was ultraviolet (UV) treated for 10 min. The gels were hydrated with PBS before culturing the cells. Matrigel (Corning) was thawed overnight at 4°C, and depending on the lot concentration, it was diluted to 2 mg/ml with cold RPMI media. The gel was dispensed in the culture well and incubated at 37°C before seeding the cells.
Peripheral blood monocyte isolation and macrophage differentiation
Monocytes were isolated from whole blood by density centrifugation using lymphocyte separation media (MP Biomedicals, Santa Ana, CA). Whole blood was collected at the Institute for Clinical and Translational Science at the University of California, Irvine, through approved protocols with the UCI Institutional Review Board, and informed consent was obtained from all donors. Monocytes were enriched by counterflow elutriation, as previously described (40), and the purity was assessed after every isolation by staining for CD11b+ and CD3−CD20−CD56. Monocytes were differentiated into macrophages with recombinant human M-CSF (25 ng/ml; PeproTech, NJ) for 7 days, with the addition of fresh media with M-CSF at day 3. Human monocyte-derived macrophages were used for experiments on day 7 of culture.
THP-1 and U937 cell culture
The monocytic cell lines THP-1 and U937 were obtained from the American Type Culture Collection and cultured according to the manufacturer’s recommendation. Both the cell lines were differentiated for 18 to 42 hours with 20 nM PMA before experiments.
Preparation of Polyacrylamide Hydrogels (PAA Gels)
Gels were produced on aminosilanized glass coverslips. Briefly, 13 mm round glass coverslips were washed with 0.1 M NaOH for 15 min, washed with ethanol and water and dried. They were incubated for 20 min in a solution of 0.1% (v/v) allyltrichlorosilane and 0.1% (v/v) triethylamine diluted in chloroform, washed and dried. Finally, they were covered with 0.5% glutaraldehyde for 30 min, washed and dried. To obtain hydrogels with a range of different stiffness, acrylamide (AA) and N,N′-methylenebisacrylamide (BisA) were pre-mixed in different proportions. For compliant gels we used 5% AA and 0.07% BisA; for intermediate 12% AA and 0.2% BisA; and for stiff 18% AA and 0.4% BisA (all v/v and dissolved in PBS). Tetramethylethylenediamine (TEMED) was added to the pre-mixes to a final concentration of 0.3% (v/v). The mixture was degassed for 10 min. We used custom-made methylsulfone acrylate (MS) monomers as a thiol-reactive compound for functionalization with adhesion molecules, as recently described in Farrukh et al., 2016. The MS monomers are incorporated to the AA/BisA mix and after the gels are polymerized they can react with the thiol group present at the Cys residue of the employed adhesion peptide. This strategy enables the uncoupling of polyacrylamide stiffness from adhesion ligand density, ensuring a comparable density of peptides between different hydrogels. MS monomers were dissolved at 32 mg/ml in dimethylformamide (DMF). To initiate the polymerization, a final mixture 1:8 (v/v) of MS monomers and the AA/BisA pre-mixes was prepared and ammonium persulfate (APS) was added to a final concentration of 0.1% (w/v). 9.3 μl of the solution were placed between a glass coverslip and a flexible hydrophobic polyester sheet to gel for 30 min. Polymerized hydrogels were peeled off, immersed in water in a 24-well plate to swell for a minimum of 1 h, washed, UV-sterilized for 30 min and washed again. Hydrogels were functionalized with 0.5 mg/ml of cRGD-Phe-Cys (Pepnet #PCI-3686-PI) diluted in ddH2O at room temperature overnight. They were finally washed and then stored in water at 4°C for a maximum of 14 days until they were used for either mechanical characterization or cell culture. All chemicals mentioned were from Sigma-Aldrich unless specified.
Mechanical Characterization of PAA Hydrogels by Atomic Force Microscopy (AFM)
Hydrogels bound to the glass coverslips were mounted on a 35 mm dish using vacuum grease and covered with PBS at room temperature. The characterization was performed with a Nanowizard 4 (JPK Instruments) using cantilevers (arrow T1, Nanoworld) equipped with 5 μm diameter polystyrene beads (microParticles GmbH) and calibrated with the thermal noise method. Gels were probed in liquid with an indentation speed of 5 μm/s and a relative force setpoint ranging from 0.6 to 8 nN to achieve comparable indentation depths of approximately 2 μm. The obtained force-distance curves were analyzed using the JPK data processing software. Parts of the curves corresponding to the first 2 μm of indentation depth were fitted using the Hertz/Sneddon model for a spherical indenter and Poisson ratio of 0.5 was assumed (Hertz, 1881; Sneddon, 1965).
Macrophage Cell Culture, Stimulation, and Inhibitors
Primary bone marrow-derived macrophages (BMDMs) were produced by cultivating bone marrow harvested from C57BL/6J young mice (Janvier Labs; ethics approval number DD24.1-5131/396/9, Landesdirektion Sachsen) in BMDM medium consisting of high glucose DMEM + GlutaMAX (Thermo Fisher Scientific), 10% heat-inactivated FBS (v/v; Thermo Fisher Scientific), 1% penicillin-streptomycin (v/v; Thermo Fisher Scientific) and 20% L929 conditioned media (v/v) on CorningTM not TC-treated petri dishes (Sigma-Aldrich) for 6 days. Differentiated BMDMs were detached, seeded on hydrogels within a 24-well plate format at the concentration specified for each experimental approach and cultured for 14–18 h. For LPS priming, cells were challenged with 100 ng/ml ultrapure LPS from Escherichia coli (InvivoGen) for 4.5 h for most of the experiments and for 6 h for the gene expression experiments. For nigericin stimulation, BMDMs were treated with 10 μM nigericin (InvivoGen) for 1.5 h. For inhibitor experiments, 0.06% DMSO (v/v; Sigma-Aldrich), 10 μM blebbistatin (Sigma-Adlrich #B0560) or 10 μM Y-27632 (Sigma-Aldrich #Y0503) were used. BMDMs were pre-treated with the inhibitors for 1 h, and the inhibitors were kept in the medium during the subsequent priming with LPS priming and stimulation with nigericin.
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I am planning to carry out gentamicin protection assay to study the internalization of E.coli in macrophages and dendritic cells. I want to specifically look at the degradation of E.coli in lysosomes. Does anyone have an idea that how long does it take for E.coli to reach lysosomes in RAW cells and primary macrophages and dendritic cells from mice?
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Hi,
I am not sure about dendritic cells, but primary macrophages such as peritoneal macrophages or bone marrow-derived macrophages need only a few minutes to take up E. coli or L. monocytogenes. We analyzed uptake kinetics of different bacteria by IF microscopy and by FACS and we observe that already 15 min after infection, about 80 % of L.m. or E.coli are intracellular.
To be sure you can add your gentamycin to the cells 1 h after infection. At this time point almost all bacteria should be internalized. I would suggest to use an MOI between 0.5 - 2, not higher.
We analyze the phago-lysosomal degradation by two different methods, either by IF microscopy (co-localization of lysosomal fluid phase markers with the internalized bacteria) or by analysis of acid hydrolase activity in a plate reader (by coating the bacteria with probes that become fluorescent upon cleavage by acid hydrolases). Here we observe that 15 min after infection about 60 % of internalized bacteria co-localize with lysosomal fluid phase markers. The fluorescence signal in the plate reader, which represents acid hydrolase activity, starts to increase about 30 min after infection and reaches a plateau about 2-3 h after infection.
To get an idea, whether your E.coli have been degraded, you can lyse your cells and plate the lysates on ager plates at different time points after infection to detect living bacteria by colony formation (CFU assay). 5 h after infection, there should be only a few colonies left in comparison to your starting point. 24 h after infection, I would expect to have no colony formation at all, since the E.coli should be completely degraded at this time point.
For further details and protocols, you can have a look at our recent studies, where we used the above described methods:
The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes. Cell Host & Microbe, (2018)
DOI: 10.1016/j.chom.2018.01.018
Macrophages target Listeria monocytogenes by two discrete non-canonical autophagy pathways.
Autophagy, (2021)
DOI: 10.1080/15548627.2021.1969765
Kind regards,
Alexander Gluschko
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Even if I originally try to seed the wells of a plate with what should be enough cells to completely cover the bottom of the well confluently, when I check them 48 hours later only a small percentage actually seems to have differentiated and adhered. Has my PMA just gone off, or is there a specific brand of PMA that you would recommend? Are there more effective ways to differentiate THP-1s into macrophages? Thank you for any input!
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Hi,
Here in our lab, we differentiate THP-1 with PMA from Sigma (P1585). I had the same problem twice. I suppose my problem was just my PMA that were gone, because I tried again with a brand new and cells differentiated well. But the second time it happened I was using also a brand new and cells did not differentiate. Once again, I tried with a brand new and it worked.
Maybe you are having the same problem. We have been working with this for so long and it happend just twice, that s why we never tried another brand.
Hope this helps!
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I am interested in culturing macrophages isolated from mice skin tissue and culture them on glass slides for staining and imaging.
Do these tissue macrophages adhere in the same fashion as like peritoneal macrophages?
if not, should they be cultured for longer time for proper adhesion?
how long these macrophages can be maintained in vitro?
what culturing conditions are recommended?
I really appreciate your help.
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Macrophages adheres the surface in 24h in humidified CO2 incubator
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I would like to know whats the concentration of lysozyme and H2O2 in the phagolysosome of THP-1 macrophage cells or in general human macrophage cells. Any idea?
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Hi,
The concentration of H2O2 in the phagolysosome depends on your stimulus (the phagocytosed particle). Without phagocytosis, you will have only a low amount of background H2O2 production.
So, if you want to analyze the phagolysosomes in your THP-1 cells, you need a particle e.g. latex beads or better zymosan particles, or some microorganisms like bacteria or fungi, which should be phagocytosed by your cells.
For simplicity, I would use zymosan particles, which in contrast to latex beads should activate some pro-inflammatory responses in your THP-1 cells, including production of H2O2.
In order to analyze the production of ROS e.g. H2O2 in a specific cellular compartment such as the phagolysosome, it is important to choose an adequate, non-diffusable ROS probe. In the ideal case, you should couple your ROS probe directly onto your particle of choice. The probe will start to fluoresce upon oxidation inside the phagolysosome and you can detect and quantify the fluorescence signal in a plate reader.
For further information, or technical details about ROS measurements, I would like to highly recommend our paper, which focuses on ROS measurements in different cellular compartments of phagocytic cells:
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling
For theoretical background, I would like to recommend an excellent review "Functions of ROS in macrophages and antimicrobial immunity" by my colleague Marc Herb. Here he gives an overview about ROS and their different sources in macrophages, summarizes the roles of ROS in antimicrobial immune defense and provides an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers.
If you like, please have a look at:
Functions of ROS in Macrophages and Antimicrobial Immunity February 2021, Antioxidants 10(2):313 DOI: https://doi.org/10.3390/antiox10020313
Kind regards,
Alex
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This is for a manuscript we hope to submit soon. Thanks
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My question was aimed at knowing if there were any standard colouring rules for the different macrophage subsets (M1 and all the M2s) in illustration, since we're preparing a figure for a manuscript. Thanks anyway for taking the time to answer Mxolisi justice Ndlovu
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I bought human CD14 + monocytes from mobilized blood (10 million cells) to generate M1 and M2 macrophages. After thawing the cells, I wonder if I can expand them (1 passage) to make stocks before starting induction. I plan to use RPMI 1640, 10% FBS, 1% P / S. Thank you!
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Hello,
I suppose monocytes are sensitive to freezing. Although on thawing they may be viable, they may lose some of their functions.
I suggest you freeze human PBMCs, and when you require monocytes you can thaw PBMCs and isolate monocytes using CD14 magnetic beads.
Best.
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Dear all,
I am doing western blot for caspase-1 P20 or P10 (active form) in macrophages in response to LPS and ATP.
According to the previous data (attached figure) and discussion https://www.researchgate.net/post/Whats_the_best_way_to_detect_activated_caspase-1_from_dendritic_cells
it seems that p20 and p10 forms are detectable in cell lysates at earlier timepoints, the bulk of these forms are secreted and are detected in cell culture medium or supernatants upon precipitation of proteins. Therefore, my questions are:
① have you measured the active form of Caspase-1? They are in the supernatant or cell lysates?
② Can someone give me a protocol of how to extract the sample from cell supernatant for future western blot?
③ Is there antibody specific for Caspase-1 P10 or P20, or we just use antibody for pro-caspase-1 and find the band at 10 and 20 kDa position.
Much appreciated.