Microglia - Science topic

If you are aware of the fact that cancer patients treated with standard chemotherapy over the past 20 or so years have cognitive problems with reasoning and motor skills, I'd like your input on why, and why ten to twenty years later.  Microglia (brain macrophages, which is a misnomer) can initiate the process of neurodegeneration, which leads to ex-cancer patients, who've survived through chemotherapy, down the path of significant brain damage.  There is an initial insult due to systemic inflammation, but I'm thinking that endothealial damage and their ability to secrete inflammatory lymphokines perpetuate or exhasterbate chemotherapy-induced cognitive problems in cancer patients.

What do you think?

Michael

Hi,

I have been growing BV-2 microglia in RPMI 1640 supplemented with L-glutamine, 10% FBS and 1% penstrep and 1% sodium pyruvate. However, it has not been growing very well recently and as the cell either die or grow slowly. I tried using fresh products but it has not helped.

I would appreciate any help you would be able to provide.

Also, I thought the BV-2 cells was 24 hours and just wanted to make sure this is accurate.

I am a little confused about the nomenclature of the cell line referred to in the citations of ATCC -CRL 2467 as the BV-2 cell line. The BV-2 cell-line as described in ACCEGEN website is derived from C57BL/6 mouse. The CRL-2467 (EOC 2 in ATCC website) is from a C3H/Hej mouse. Can someone working with microglia cell-line clarify this.

Thanks

I digested brain tissue with 0.25% trypsin at 37°C for 30 minutes, then isolated microglial cells using 30% Percoll gradient centrifugation. I cultured the cells in 5% MEM or DMEM medium, but the cells barely adhered. Under the microscope, the adherent cells appear as small, round dots and lack the typical morphology of microglial cells. I also tried coating the plates with P-L-L, there was no improvement. I confirmed the cell phenotype using flow cytometry, identifying them as CD11b+CD45int and CD11c+. What could be the reason?

hi everyone

I am having trouble in transfecting microglia N9 cell line. Now i decided to use Nepagene electroporator to transfect these cells. Can anyone send me a protocol for this as i am new to this.

I am researching the microglia related neuroinflammation in CNS, and want to know the earliest time of microglia after acute inflammatory stimulation.

I am trying to grow microglia culture for 10 days and after day 2/3 there are certain flat non-microglial contaminating cells observed. They are rapidly growing and some are even growing on top or in contact with microglia. I am trying to find a way to distinguish what these cells are and wanted to know if there are any ways of determining these cells? Could it potentially be fibroblasts or mesenchymal cells?

I have noticed that, regardless of whether isolating primary microglia from humans or mice, purity is generally assessed using CD11c flow cytometry staining. Why not use CD11b instead?

Hello, I am checking the inflammatory reaction that occurs when LPS is treated on BV2 microglia. When I treated LPS on BV2 cells, I confirmed a noticeable increase in IL-6 and I expected that JAK2 would be phosphorylated by LPS considering IL-6/JAK2/STAT3 axis, so I checked it through WB. However, I can't see the WB band at all. I can see total-JAK2, but I can't see the band at all on phospho-JAK2. Strangely, STAT3, the sub-signal of JAK2, was confirmed to be phosphorylated by LPS. But why can't JAK2 be confirmed.. Please help...

I am working with microglia SIMA9 and I will perform a qPCR. If you have already worked with them, can you suggest me one?

What are the appropriate markers to detect resting microglia? Studies have reported IBA-1 which is also extensively reported in M1 polarization. Some other studies have reported CD11b and CD45 as well but those are also found in activated microglia. So is there any certain marker to check resting microglial state?

Dear colleagues, I'm planning to start neuron mouse co-culture with astrocyte and microglia but I heard that it's difficult to keep it up. Any advices?

I was wondering if anyone knows an antibody that can be used for staining microglia in zebrafish? I am familiar with 4c4 and L-plastin antibodies, but these are not perfect for me. Is anyone familair with, for example, a P2Y12 ab that works in zebrafish ?

Thanks!

I am currently learning to culture primary microglia cells from P0-P2-day-old pups, and after 14 days of culturing, I got these cells. I performed IBA1 staining to check purity, but still, I am worried since when I performed the seahorse experiment, irrespective of the cell seeding density, I was getting very low signals.

I would need a human microglia cell line Cirspr-edited KO for TREM2. I am pretty sure to have read a paper with HMC3 trem2 KO but I cannot find it again!! Any help??

What is the immunological test through which we determine the activation of microglia cells?

Hello!

We are generating conditional knockout mice using CX3CR1 Cre for a particular gene. During this process, we are performing tail cuts for genomic extraction and genotyping. We've designed primers for flox PCR to detect a deletion band if Cre is present. However, we've encountered an issue. Despite the tail typically having fewer cells like those containing CX3CR1 Cre (such as microglia, macrophages, dendritic cells), deletion bands are occasionally detected in tail genotyping. In some mice, these bands appear stronger than the normal flox allele bands. Conversely, in other mice with Cre, no deletion bands are observed at all. Could this be due to low-level expression of CX3CR1 in some cells in the tail? Should germline depletion be considered in these scenarios?

Thanks,

Hello!

We know of the traditional ways to isolate microglia from primary mixed cultures using shaking and tapping. However, these microglia are not very healthy and cause inconsistency in the reproducibility of experiments.

We are aware of methods to obtain primary astrocytes using clodronate or trichostatin-A. Is there a similar way to obtain pure microglia from a mixed glial population?

I shake microglia (200rpm, 2h) from P0-P3 mice after 12-14d culture of primary mixed glia. After another 3-4 days culture, I stain the cells with Iba1/GFAP/Oligo2 antibody, but I find that all these markers can stain every cell. Has anyone encountered this? What is the possible reason?

My IF protocol: 1. wash three times with PBS, 2. fix cells with 4%PFA and 120nM surcose in PBS for 15min at RT, 3. 3 x 5min wash in PBS, 4. block cells with 3% donkey serum, 5. incubate cells with 1 antibody over night at 4℃, 6. 3 x 5min wash in PBS, 7. incubate cells in 2 antibody for 1h at RT, 8. 3 x 5min wash in PBS.

I need to quantify microglia and astrocytes through immunofluorescence, however I would like to know the best way to quantify, whether fluorescence intensity or manual counting or some other type

I am trying to stain lipid droplets in microglia in the RPE flat mount and I was wondering the protocol and dilution to do so. Anything would be appreciated!

Hello Everyone !

I isolated mixed cells from neonatal mouse cortices , and they are always contaminated with other cells (by imaging it appears that my astrocytes are even the least common population) .. any ideas what I can do to prevent this ?

I plate on pdl coated flasks , and shake the mixed cells when confluent (1 hr on 180 rpm to remove microglia) and (4-6 hrs on 250 rpm to remove opcs)

I have been working to troubleshoot my Iba1 stain for microglia for the past few months with no avail. Looking to stain Iba1+ microglia in fresh frozen, OCT-embedded rhesus macaque brain, slices 10 um thick on microscope slides.

Conditions already used:

Fixation solution of 63% PIPES buffer + 37% PFA (4% stock) OR fix at 4% PFA alone

Fix for 10 minutes at RT, block with Donkey block (10% NDS + 0.1% Triton + 0.01% NaN₃)

Primary antibody incubation

Dilutions used: 1:100, 1:250, 1:500, all attempted both 1 hour at room temp or 4 degrees overnight

I utilize the same protocol for all of my other antibodies and haven't had an issue, but this one just keeps escaping me. Does anyone have any pointers for fresh frozen macaque brains? Most of the literature I find on Iba1 staining is for FFPE or previously fixed brain tissue. I have tried a few different antibodies from different manufacturers (Novus, Sigma), but none have been remarkably successful at giving me the level of staining I am expecting to have.

Thank you!

How much do neutrophils, microglia, and other immune cell activation and infiltration play a role after craniocerebral trauma?

I have this problem for sometime that Microglia cells in these PLL coated wells are dying after sometime cause they don't adhere to the coverglasses, I've tried different strategies and it shouldn't be a complicated process but still nothing :(

Hi All,

I am working on microglia dynamics in vivo with two photon microscope,but I cannot find a method to mesure microglia process length chang,I have tried imageJ plugin MTrackJ,but it looks work only on particles.Can anyone propose some methods to work on?

I have attached the microglia morpholoy,red-retractions,green-extensions.

Thanks in advance.

I get primary microglia from P0 transgenetic pups with flox. after 10-14 days mixed culture, I get microglia and seed on 12 plates. I want to knockdown the specific gene with flox, then I used a lentivirus (pultra-cre) which my coworkers have proved can upregulate microglia gene with stop flox. But it dose not work on my microglia. I extract RNA and stain cre/Iba1/GFP to verify the knockdown effect (as the figures show, it dosen't work).

I wonder if someone has met this before. Why a lentivirus can be used to upregulate one gene with stop flox but can not be used to downregulate another gene? I used the same DMEM and FBS as my coworkers.

We are seeking to count cell death stain from confocal image stacks; however, Neurolucida is quit expensive. Are there any alternatives that you recommend? Is ImageJ viable?

I want to study the expression of some proteins related to oxidative stress with western blot. I wonder what microglia cell line is the best for it.

Dear all,

I am looking to do FACS sorting from a copopulation of BV2 and RAW 264.7 cells back to individual populations after LPS or alpha-synuclein challenge. I understand that they are both macrophage-derived cells and am having issues finding a unique expression marker for each population. Do you have any good strategy for FACS sorting for the cells?

Many thanks and kind regards,

We only tried lipo3000 and lipo6000, but the transfection efficiency was not good.

Hi all, we met a problem that we follow the protocol from the company and successfully have the ipsc-derived hematopoietic cells with reasonable population. But when we start the microglia differentiation steps, the cells stopped dividing, and the population got decreased. The microglia-like cell looks good under microscopes (shapes, branches), however, because we can't get enough cell ,we can't test the biomarker and can't do anything next.

Is anyone know how to solve this problem, or how to increase the population during the microglia stage?

Hello everyone! I am trying to find a protocol to isolate microglia from mouse embryos. (the mouse model I use produces homozygous lethal pups) All of the protocols I have read are for adult mouse brains. Does anyone know or have a protocol for younger mice?

We would like to use LPS for microglial activation (M1 polarization). These are iPSC-derived microglia and are relatively less sensitive compared to monocytes or macrophages.

I see a lot of different LPS strains available through the Sigma Aldrich website:

O111:B4

O26:B6,

O55:B5,

O127:B8,

O128:B12

1. Would anyone happen to know how these LPS strains compare in potency? Which strain is most potent for inducing M1 state?

2. In my reading, O111 and O55 are the most commonly used strains for M1 polarozation? Does anyone know how they compare in potency?

Thank you.

I am quantifying microglia using ImageJ and focusing on the circularity and density parameters of these cells in the olfactory bulb of mice that have been inoculated intranasally with VSV at 2,4,6, and 10 DPI and am having those compared side-by-side with mocks to comment on the inflammatory state of these mice characterized by the microglial response. I read that reactive or inflammatory microglia grow in size (which is something I see consistently across all DPIs), proliferate, and retract their processes and hence become more circular and dense to accommodate for their phagocytic functions. The increase in both size and number is significant (figures not attached), but I am not entirely sure how to interpret the circularity and solidity (or density) results I have in this dataset. Is it known for microglia to adopt specific phenotype/s directly post infection and change throughout the infectious course? I am essentially trying to understand the reason analyzed microglia are dropping in circularity and density on days 4 and 10, but not exactly days 2 and 6. I would have expected the circularity and density to rise across all days or at least climax at days 4 and 6, given that that's when they are the most sick of this virus. I would appreciate any help I could get or referencing of info I can read on this topic to comprehend whether these phenotypic changes translate into different functional roles, if at all.

Hi everyone.

I have a batch of microglial cells differentiated from PBMCs that I want to use for a migration/invasion assay when exposed to some chemotactic cues. I am trying to use the Boyden Chamber (aka Transwell) system. However, since I need to differentiate these PBMCs in microglia for 10-14 days and they die if I attempt to split them, I have to plate them directly inside the transwell and keep them for very long in culture. I cannot add media into the plate well, as cells may migrate towards the external side of the transwell membrane during their differentiation.

I'm trying to isolate EVs from primary microglia and I'm getting low concentrations so I want to stimulate them with ATP, because it was shown to increase EVs secretion. Do I need to dissolve the ATP in a certain solution (some papers used it with KRH buffer)? Can I add it to the medium and then wash and add fresh medium? And for how long do I need to let the cells secrete EVs after stimulation?

I would appreciate any help with this issue

Thanks in advance,

Orit

Hello all,

For my upcoming experiment, I need a suitable positive control for the establishment of my iNOS (NOS 2) primers. I would like to study iNOS expression on mRNA level at different time points in microglia and astrocytes from rats. Maybe someone can help me further, I would appreciate it very much! Thanks a lot!

Can anyone tell the difference between microglia, astrocytes, and even oligodendrocytes? Here are the cells shaken off on day 20 of the mixed glial culture method. I wanna get pure microglia but it seems failed.

The first picture is the mixed glial culture DIV20 days. The second picture is the cells shaken off form manually the first picture mannually.

I've observed that microglia cells in a mixed culture grow and proliferate better compared to pure microglia culture even though countless papers talk about culturing pure microglia.

Could anyone shed some light on this.

Media used: DMEM(F12) + Glutamate, supplemented with 10% FBS & 1% P/S and one time only 5ng/ml GM-CSF.

I do not use Percoll isolation, I exploit microglia adherent property for my isolation.

I've just started working with HMC3. Bought from ATTC, using MEM with 10% FBS 1%P/S. I split them last week when they had reached 80%. They do not seem to be growing but for the most part look healthy, although I do see some floating cells. I was told not to change media as often as the ATTC guidelines suggest, will this slow down division?

Any help would be great!

I am currently generating two AAV2/6 and 2/9 'human CD68 promoter: GFP-Myc-NLS (SV40)-Cre".

For AAV production, I transfected DNA into HEK cells.

HEK cells well expressed GFP-Myc-NLS (SV40)-Cre and I can see the GFP signal localized in the nucleus.

But, I can not see any microglia that do not express GFP both in vitro and in vivo.

(AAV2/6 titration: 1 x 10^11 vg/mL, AAV2/6 titration: 1 x 10^14 vg/mL)

Why my experiment failed?

Promoter and AAV2 backbone from Addgene #75033

GFP-Myc-NLS (SV40)-Cre ORF from Addgene #105540

Hi! I am looking for a human microglia cell line that was derived from an adult, preferably brain tumor patient or "healthy" individual (accident/post mortem). Can you recommend any / do you know of any? Human microglial lines are scarce in general, and the ones I came across were fetal, could not find adult / elderly one so far. Thanks!

Can anyone recommend a good marker for activated microglia? I am hoping to FAC sort microglia and am looking for a good extra-cellular marker to further separate my cd11b labelled cells.

I can't seem to find any literature on the expression of MAO-B in microglia.

When detecting the activated microglia, studied commonly use the increased expression level of IBA-1 in IF or IHC to show the activation of microglia, since the proliferation of microglia is dependent of activation of microglia, why not use calculate the number of activated microglia or use both which seems more convincing.

Hi all,

I've been having issues with staining my postnatal day 7 rat hippocampal and lateral ventricle slices for iba1. They are 5µm thick, so quite thin. However, my postnatal day 21 rat slices are also 5µm thick and iba1 looks great on these slices.

According to the literature, there should be staining present in both of these areas so I'm wondering if it's worth either using a different secondary antibody or if the slices are just too small and thin at that age? I would love to hear any thoughts on what steps to take next.

- Paraffin-embedded tissue sliced on microtome (5µm thick)

- 4% paraformaldehyde and PBS used for perfusions, post-fixed in 4% PFA for 24 hrs then soaked in 70% ethanol

- Coronal sections

- WAKO anti-rabbit iba1 primary AB

- Donkey anti-rabbit 555 (thermofisher) secondary AB

Thank you in advance,

Melissa

Does anyone ever done cell sorting of microglia from 1 whole mouse brain? how many total cells do you obtain and how many specific cd11b after cell sorting?

What about 2 hippocampi?

I'm trying to isolate microglia cells from adult mouse brain and from specific area such as hippocampus and i'm interested to understand how many cells I can get from the total and after cell sorting!

Thanks all :) :)

Elena

I am trying to polarize mouse microglia to M1/M2 using LPS/IL-4.

I would like to use immunocytology to confirm the polarization, and I would like to know if you know of any antibodies that have fewer false positives.

Currently, I am using CD68 and Arg-1 (Santa Cruz).

These antibodies are positive for both M1 and M2.

Thank you very much in advance for your suggestions.

I am culturing iPSC-derived as well as primary rat neonatal and adult microglia on collagen IV-coated TC ware. I wanted to perform some flow cytometry experiments and tried detaching cells from the surface with Accutase (40 min incubation at 37C, as described in Reich et al https://www.frontiersin.org/articles/10.3389/fimmu.2020.617860/full#h3), however microglia are still stuck to the surface and even quite aggressive tapping of the well plate does not facilitate their detachment.

Has anybody else faced the same issue and could advise on how to detach microglia from the surface?

Thank you in advance!

Hello dear scientists.

I have a question regarding the EOC-20 microglia cell line. I will perform cell culture with this cell line and then ICC for the first time. I do not have a proper protocol, and I've searched and read a lot but can't find a detailed protocol in that regard. for example, I need to have a culture schedule that explains when I have to split cells and so on...

I appreciate any help in this regard.

Thank you a lot in advance.

I am trying to culture primary microglia cells from adult mice but for some reason I am getting really small cells and not seeing the typical morphology of microglia cells. Even when cells are put in MCSF media and left for a couple days I am not seeing any change in their morphology or any adhesion to the plate. Could someone please let me know if there is something I can do to get better results?

Dear all,

I'm searching a validated protocol to obtain microglia cells from iPSC.

Thanks

deleted

Hi all,

I am culturing primary microglia and using M-CSF for their expansion. Up until now, we have been producing our own M-CSF from L929 M-CSF O. E. cell line, but due to high production costs and laborious procedure we are considering buying purified M-CSF, also to reduce inter-experiment variability.

I have found conflicting information about the dose of M-CSF to use for primary microglia expansion, and I have little way of knowing how much M-CSF I was using, as it is secreted in the medium and we would use the conditioned medium as it is.

Microglia people, how much purified murine M-CSF do you use in the growth medium for your primary microglia?

Thanks in advance!

Hi everyone,

I'm working with primary mouse microglia, and I'm looking for a primary antibody that I can use as a microglial marker for resting and active microglia in ICC. Our lab usually uses the WAKO Iba1 antibody, but I need to costain for cannabinoid receptor 2, which is also a rabbit antibody. I'm looking for a marker that is mouse-reactive but is not derived from a rabbit host.

Does anyone have any suggestions for targets I could use?

Thank you in advance!

Hell, I am isolating microglia cells from adult mice, using Miltenyi magnetic beads. I want to perform experiments with these Microglia cells but I don't know how long I should wait till I able to do so. If some could please help me out I would be grateful.

I'm currently working on evaluating the proliferating property of retinal microglia. What makes me wonder for a while is the differences between the two strategies: cumulative labeling and pulse-chase labeling.

For example, what are the differences between giving the mice repeated doses of EdU/BrdU daily then evaluating the microglia proliferation (i.e: on 1st/3rd/5th/7th days after treatment) and giving the mice a single dose at 1st-3rd-5th-7th day then collecting the sample for evaluation?

Thank you in advance.

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.

I am trying to measure microglia activation in response to an insult with qPCR. I have used Iba1 and found no differences in the expression between my groups at different timepoints. I am using a developmental model where the insult occurs at postnatal day 10. We have found increase in MhC II+ cells in the same model using flow cytometry which show that there is microglia activation... now I am trying to look for other markers of microglia activation with qPCR. As there was no difference with Iba1, I was wondering if maybe there was another marker that could show differences since during development microglia can present different populations. I have seen a talk about differences in microglia population measured by Iba1+ and P2Y12 + cells. So, I am wondering if measuring P2Y12 expression could be an interesting approach. Any ideas?

Hello, I'm looking for an anti-TLR4 antibody for IF in human microglia in culture.

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

My lab is trying to quantify the number of microglia and we were using PU1, but we have been having issues with it not being very good (the staining isn't very strong, a lot of background, etc.). My lab has decided to try out another antibody, but most antibodies that label for microglia label the processes as well and that would make it hard to quantify the total number of antibody in a region.

Dear all,

I usually use cell media with phenol red. However, I need to use media without phenol red since I need to measure fluorescence from the media. As far as I am concerned, there has only been reported that phenol red has some estrogenic effect in MCF7 cancer cells. But nothing has been reported about the effects of phenol red in brain metastatic cell lines and in microglia or other brain resident cells. If you know something about it, please let me know.

I have also been looking around if there are any differences between media with and without phenol red. Indeed, there are some and I was wondering if any of you could tell me what they are due to or if these changes can affect my experimental results. Here I spot the differences.

- Whereas in DMEM with phenol, L-Tyrosine is used, in DMEM without phenol L-Tyrosine disodium salt dihydrate is used. The concentration of these aminoacids is also different.

-Sodium Pyruvate is present in DMEM with phenol red and absent in DMEM without phenol red.

-The final concentration of CaCl2-2H20, MgSO4-7H2O and NaH2PO4-H20 is different in these media. 

Moreover, we are about to buy a cell line from ATCC and they recommend us to use their DMEM media. I think I will use DMEM media from Gibco since this is the one we currently use and we have. Do you think this can affect cell growth or our results?

Thank you in advance,

Anna

I am looking for optimal software to count microglia in the human brain.

I would like to do a co-culture of microglia and astrocytes for making a model of spinal cord inflammation. We have some concerns regarding compatibility between them. We want to be sure they can grow together in a culture.

Can anybody suggest good microglial and astrocytes cell lines for that?

I will mark tissue from animals treated with LPS and found many different markers, such as CD11b, CD68, MHCII, CD40 and CD86, for microglia activation. Does anyone know which one might give me the best results and is more specific?

Hello. I intend to simulate extracellular epileptogenesis-like conditions in microglia cultures. I concluded that the most well-fundamented and ready available way is to alter ionic concentrations. I decided to start with increasing extracellular potassium concentration to 10-12 mM.

For the in vitro model, I use RPMI 1640 glutamax medium, which already contains KCl 5.333 mM.

I intend to incubate the cells for a period with high K+ cocentration, incubate with normal medium once again and continue the experiment for a number of days more.

What would be the most correct way to increase the potassium concentration from 5.333 to 10-12 mM in this readymade medium?

Thank you for the attention

Gentlemen,

We are preparing a project to access a grant, but we are missing a lab that can help in the first phase of Proof of Concept and Safety.

The lab should be European (But not spanish, and sorry, but not UK) and be seasoned in brain samples handling, and also if possible having experience in cell morphology study (Neuron, Microglia, Astrocytes) or metabolic study (on said samples)

The project includes the development of a protocol in humans but we need to try it before on brain samples, and we are in a hurry to find a lab that can provide experience in this field.

Since we are in a hurry, you might contact me directly or respond in the thread.

Sincerely, J Vigil

Im trying to find an available software that can help me quantify the process thickness of microglia in images I have. I have previously used NeuronJ through Image J to measure their length, but I do not believe I can measure thickness through this method. Is anyone aware of a software package that can manage this?

I am attempting to design a high throughput FACS based phagocytosis assay to investigate the effects of a couple of different drugs on the rate of phagocytosis in a stimulated immortalized microglia like cell line. I know the standard of the field for the FACS based assays is to conjugate the substrate that people hope to be phagocytosed to a PhRodo dye to ensure that the signal is composed of only internalized particles versus objects which are just lodged in the membrane, but I am not sure how the dye would interact with the conjugated protein. Does anybody have any experience with these molecules? Any tips or concerns? Thank you in advance!

Hi all,

I need to learn how to use ImageJ to do colocalization and quantification of my IHC mice brain slides.

I have mice brain sections that I stained with the microglial specific marker iba1, and costained it with M1 and M2 markers iNOS and Arginase-1, with DAPI as a counterstain.

So my slides are as follows:

DAPI

iNOS --> Alexa Flour 488

Iba1 --> Alexa Flour 594

And

DAPI

Arginase 1 --> AF488

Iba1 --> AF 594

I couldn't find a resource on how to quantify this, any help would be appreciated.

I have seen many papers and discussions here about various techniques for isolation and culture of microglia and astrocytes from adult mouse brain. Isolation of these cells by Miltenyi magnetic beads is supposed to yield a great purity and have been used for short-term culture (6-24h?) and RNA analysis. Can anyone give me feedback from their experience about separation of cells using Miltenyi beads and culturing these cells for a week (or at least up to 72h) , as described in the Miltenyi protocol?

My lab is now far past when we should have microglia in our 24-well cultures. We have plenty of what we assume to be astrocytes lining the bottoms of the entire wells but we cannot seem to get microglia to start growing atop the astrocytes. Any recommendations?

- We are using GlutaMAX DMEM with 10% FBS and 1% P/S, washing the wells once every two days with 1X HBSS and replacing the media with fresh media...

- We are now over 20 preps in and we cannot successfully obtain LPS-induced nitrite concentrations from our cultures because it seems that our microglia are not growing...

-Has anybody had this problem before?

I'm planning to culture primary microglia from newborn mice. I'm not sure which method of microglia isolation. Does anyone use TrypLE Express for primary microglia isolation and culture?

Plsease tell me the yield of using TrypLE Express over mild-tripsin.

I have some murine brains and spinal cords from an EAE model that I want to process and do RNA analysis with. I want to search for differences in OL, astrocytes and microglia trancripts to see which of them are overexpressed and distinguish between their populations, but I´m not sure if it is more appropiate to focus on exctracting RNA from a specific zone of the brain or otherwise do it from the whole brain. I know that in a EAE model the main effect will be done in the spinal cord, which I will do the RNA extraction from the whole estructure, and in brain there will be less lessions. Doing it from the whole brain will dilute the results I will obtain if there is a focalized part that is affected, the problem is that I don´t know which region is that one and the literature I read is not very clear about it, some of them focus in some regions and the others process the whole brain.

Thanks a lot!

Hi everyone. I am going to isolate microglia from 2 months old mice using the Percoll gradient (30, 37 and 70%), for subsequent FACS sorting and mRNA isolation for RT-qPCR on inflammatory cytokines. This type of isolation is a relatively new technique in our lab, so I would like to ask if anyone here is doing something similar and could give me a few tips: how many brains (cortices) do you pool together? According to manufacturer, the Percoll should yield 200.000-400.000 microglia cells from 1 adult naive brain - I am new in working with microglia, but this seems to me a very optimistic number!

Also, I was told that for adult brains I can be less strict and use a 70/40% Percoll gradient, so that immune cells will be a pellet, instead of a tricky interphase band. What are your thoughts/experiences about it?

I am happy to receive any kind of advice. thanks!