Macrophage - Science topic

Could you suggest some articles?

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.

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).

Hello.

Hope be useful for you:

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391981/#:~:text=Isolate%20the%20Stromal%20Vascular%20Fraction,cells%20pelleted%20on%20the%20bottom.

2. https://bnrc.springeropen.com/articles/10.1186/s42269-022-00747-7

3. https://www.jove.com/t/50707/isolation-of-adipose-tissue-immune-cells

4. https://link.springer.com/protocol/10.1007/978-1-4939-9195-2_18

5.

6. https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.biorxiv.org/content/10.1101/336032v1.full.pdf&ved=2ahUKEwiP9YqYoa_5AhWji_0HHSQ4A7M4ChAWegQIHBAB&usg=AOvVaw2hq8TcJGN37cMZnFeGzz86

Best wishes

F. Nazari

Hello Dr. Mauricio, is there any effect of lidocaine while detaching THP1 macrophage from culture plate?

we use in BSL2 lab. You should probably contact safety office at your institution to find out what they classify them as.

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.

Thank you Giorgio Perino and Filip Konecny for your answers!

Hi Sudeep Kumar Maurya

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

Dear Sönke Weinert ,

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

Yes very possible. This article at will surely be of help.

This publication by Murray et al. (Immunity. 2014 Jul 17;41(1):14-20.) may also be useful to you:

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.

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

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?

If isolating mono-nuclear cells and then differentiating them helps, here is a protocol.

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.

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.

Ikemefuna Ikesee limited in what way?

Hello Batoul Shunnar

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.

Sudeep Kumar Maurya thanks a lot for your support!

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!

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).

Visit also the following useful RG link:

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.

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)

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)

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!

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

Follow this link, you can take some information from this paper regarding CAR.

Hello Yichun Zhang

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.

By Steven D. Douglas, Steven H. Zuckerman and Samuel K. Ackerman

Pages 33-42.

Best Wishes.

Yeah, I was thinking about this. Will try both approaches.

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.

A common method is to dissolve the membranes containing the proteins with a non-denaturing detergent, such as dodecylmaltoside, octylglucoside, and some others.

Please go through the link -https://www.google.com/url?sa=t&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwjdoq-mxfz2AhXkR2wGHcb6BrUQFnoECAQQAQ&url=https%3A%2F%2Fwww.frontiersin.org%2Farticles%2F10.3389%2Ffimmu.2020.01248%2Ffull&usg=AOvVaw0A1f2Q04n9_ygMBt-FzCLe

You can use RAW 264.7, THP-1, U937

Your best option will be to use an Clec4f–DTR mice. Then you can specifically deplete KCs.

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

Chi Wut Wong

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!

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

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!

Thank you very much everyone for you suggestion.

Lora Benoit , Martin Waterfall Irin Amin Heya & Mohamad Aref Mayo

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.

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:

Sorry that I don't have an exact answer to your question yet but maybe this helps a bit :)

Best regards

Felix

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.

Two questions for you:

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.

Louise Dolan you're welcome.

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.

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:

https://youtu.be/u3dI47shZLo

Try this one.

It's easy to apply, the only thing you need is FIJI.

Based on: https://pubmed.ncbi.nlm.nih.gov/34375551/

Regards

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

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.

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.

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).

Hello Ana Lesmes Ortega

Please refer to the research paper attached. The investigators demonstrated that the culture conditions of THP-1 cells can alter their response to PMA.

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.

Thanks Nathan Kieswetter for the reply. I will try to run a pilot first and proceed accordingly.

You might try CD115 (c-fms) which is the receptor for M-CSF. I know that mouse M1 macrophage express CD115.

CRP can elevated due to any acute inflammation from bacterial causes or other causes. CBC may be change or not in Acute inflammation.

DearAhmed,

This is bacterial contamination.

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.

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.

Good luck!

Dear Melonney Patrick

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

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

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

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

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.

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.

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.

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)

Autophagy, (2021)

Kind regards,

Alexander Gluschko

Macrophages adheres the surface in 24h in humidified CO2 incubator

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

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

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.