Dear Rakesh Sharma,

This is a common problem with this method. Isolated microglia attach very firmly and is hard to detach from the plate.

Option1 is to plate mixed glial cultures in the type of plate or flask you need for your final experiment, do the mild trypsinization when mixed glial cultures are ready, and do then the experiment with your isolated microglia already on the well you want, no detaching is needed in this way.

Option2. Sometimes option 1 is not feasible (e.g. if you want to add isolated microglia on top of neurons). In this case what we do is to treat the isolated microglial cultures with Trypsin 0.25 for approximately 5 mins at 37degC and then use a cell scraper to very carefully help detaching microglial cells. It works but there is always a high mortality. The more you do it the higher the yield.

Good luck

I start with question 1:

I cannot be sure without seeing your cultures, but my first guess is that your mixed cultures may not be mature enough by the time you do the mild trypsinization step. The fact that there is no microglia below when you do the mild-tryp suggests your cultures are not mature enough. Also, the fact that astrocytes do not detach on one piece suggests also that these cultures are not mature/confluent enough. I would first try seeding at a somewhat higher density and/or leaving the primary cultures some more days in culture before doing the mild-tryp. If your primary mixed culture is dense and fully confluent then astrocytes should detach in one piece.

Also, immunocytochemistry staining for microglia would help you decide whether there is enough microglia in your primary mixed cultures.

Question 2:

We get sometimes batches of less active trypsin and then use 1:2 instead of 1:3 dilution and then the process goes faster. Also, increasing the volume of diluted trpsine per flask can accelerate the process. In any case, we have also studied whether leaving the diluted trypsin for one hour is detrimental for microglia and it is not.

Question 3:

I do not see why not and I think some authors do it. We do not. The problem is that the best moment to do shaking does not coincide with the best moment to do mild-tryp. The best moment to do shaking is when you see your cultures full of refringent microglia which is 2-3 days after confluency. The best moment to do mild-tryp is later, 7-10 days at least after confluency. So, you can do both, but if you want to mix the microglial cells obtained by both methods the final yield will not be much higher. You could do shaking, do the experiment and then wait 7-10 days and do the mild-tryp and do a second experiment. But then you will be working with microglia obtained by two different methods and they may behave differently in some ways.

Question 4 is the easiest. There is no need to coat plates when preparing mixed glial or microglial cultures. We never do it. Thanks for your questions!

Thank you Dr Josep.

Indeed, i too feel density might be an issue with my cultures. Will keep that in mind the next time i perform it. Besides, i performed mild tryp at 3-4th day after confluence in time with the shaking protocol and that settles my third question too. This time i shall exceed the time to 7-10 th day for trypsinization.

I do find a full grown layer of well differentiated, ramified to round/rod shaped/ amoeboid microglia beneath the astrocytic layer. However, at random patches even the microglia seems to be detached, and i found that out when i plated the detached astrocytic layer for further experiments. This microglial population in subcultured astrocytes is significant, and differs from the refringent microglia growing on the top in morphology and phase contrast.

An alternative that you suggested about a shaking followed by a mild tryp after 7-10 days, is in fact being carried out right now by as as curiosity based experimentation to find out the difference between the two kinds of microglia. Your explanation satiates the confusion.

It was very informative and helpful piece of information. Thanks again.

Why don't you use MACS CD11b Microbeads for obtaining Microglia?

We use CD11b microbeads to purify microglia ex-vivo from adult mice in order to analyze the microglia RNA or protein, not for culture. I think you can isolate microglia from a mixed glial culture with CD11b beads but we have not done it since the mild trypsinization works well in our hands and gives us plenty of microglia. Do you have experience with culturing microglia isolated with beads?

I have not used your method of deriving microglia- so I cannot tell whether your method is better than what we are doing. We are deriving the microglia directly from neonatal mouse cortices via the Microbeads and culture them. The cultures work fine and are suitable for staining, FACS and LPS stimulation.

This question is again addressed to Dr. Josep Saura. We did the cultures as per the outcome of the discussion. We could Indeed get the astrocyte layer completely detached. Thank you for the valuable inputs. However, we felt that microglia further harvested (using 0.25% Trypsin EDTA for 5 minutes) could not differentiate as beautifully as seen in the flask before trypsinisation, for most of them had a spherical morphology. I was wondering if it is mandatory to use Glial conditioned media for their proper sustainence and differentiation. We are using DMEM-F12 supplemented with 10% fetal bovine serum.

Thanks and regards,

Poojashri.

Thanks for the question. My first advice would be to avoid trypsinizing and replating microglia if possible, they do not like it. What we do is to plate the primary cultures in the type of plates/flasks we need for the final experiment.

In our hands microglia do not grow well in the absence of astrocytes and the mixed glial-conditioned medium is very useful to enhance survival of isolated microglia. Probably this could also be accomplished with cytokines (M-CSF? GM-CSF?). We routinely work with the mixed glial conditioned medium and work with isolated microglia as soon as possible, typically 24h after isolation.

Good luck

Hi, i am also workoing on the isolation of primary glial cells using the mild trypsinization protocol, but i fiound that the cells atttachede on the dish were unevenly. I ususually isolated the microglial on DIV 20~25, but sometimes I found the ppurified microglia were

not enough for my research, I am wondeing if you erev encountered these problems and do you have any advice?

Dear Yuan,

I too am i beginner myself. I feel the problem may be with the density of the culture, as Prof. Josep Saura also wrote in the answers.

Yield has been a problem always. But i believe the way Prof saura said, perhaps confluence of astroglial cells shall actually lead to increased microglial proliferation, and hence a better yield. But you said you do it after 20 Days DIV? In that case i donot think confluence should be the limiting factor here. I would suggest this aricle by saura et al., http://www.jneuroinflammation.com/content/4/1/26, it hlped me a lot to understand the factors contributing to microglial growth..

Hope this helps

A quick question for those that are working with this protocol.  

I plated my cells in a standard 10cm TC dish with glass coverslips  on the bottom and the cells are growing very well.  Since roughly 8DIV the cells on the coverslip have been 100% confluent but there are still patches on the plastic which are not yet confluent.

Starting at 10DIV there were some individual cells lifting which didn't surprise me given the level of confluence.  Now as of 12DIV there are clearly patches of cells which have lifted (their appearance is reminiscent of the sheet of astrocytes that I expect to trypsinize off although smaller and appears to have lifted from either the plastic section or an upper layer on the glass coverslips as the coverslips themselves continue to be 100% covered).  Is this to be expected?  Should I continue to wait for the full dish to be 100% confluent before starting to wait the 7-10 days before I trypsinize?

Thanks!  

In young cultures microglia spontaneously detach but I have never seen patches of astrocytes detaching spontaneously in young cultures, I don’t know what to think. I don’t have much experience with coverslips. I think you can get quite different results depending on the glass used. When I worked with coverslips I did polylysine coating.

I assume you are interested in having microglial cultures in your glass coverslips, therefore I  would trypsinize some 7-10 days after cultures were confluent on the coverslips, regardless of how confluent they are in the plastic (in your case this would be at DIV15-18).

Thanks for the prompt reply.  Yes my goal is to ultimately trypsinize and then use the glass coverslips for immnofluorescence to characterize the microglial cultures before proceeding with co-culture in future experiments.  I will proceed and complete the trypsinization on DIV 17-18. 

Dear Dr. Josef,

I have tried the mild trypsinization protocol from your paper, and I was very successful with the micorglia isolation. 

But I'm having problems in trypsinizing the microglia cells post the isolation. I perform the mild trypsinization protocol with DMEM:0.25%Trspin-EDTA (1:3) for about 1 hr, and then exchange with fresh media for 24 hr. And after 24 hr, I find it hard to trypsinize the microglial cells. I have tried with 0.05%Trypsin-EDTA, 0.25%Trypsin-EDTA, but they wouldn't detach.

Could you help me in this regard?

Looking forward to your reply.

Thanks and regards,

Rakesh

Dear Rakesh Sharma,

This is a common problem with this method. Isolated microglia attach very firmly and is hard to detach from the plate.

Option1 is to plate mixed glial cultures in the type of plate or flask you need for your final experiment, do the mild trypsinization when mixed glial cultures are ready, and do then the experiment with your isolated microglia already on the well you want, no detaching is needed in this way.

Option2. Sometimes option 1 is not feasible (e.g. if you want to add isolated microglia on top of neurons). In this case what we do is to treat the isolated microglial cultures with Trypsin 0.25 for approximately 5 mins at 37degC and then use a cell scraper to very carefully help detaching microglial cells. It works but there is always a high mortality. The more you do it the higher the yield.

Good luck

Dear Dr. Josef,

Thank you for the quick reply.

I would definitely try the Option 2 you have suggested, as this suits to my experimental conditions. 

As a suggestion, do you think using cell lifters, instead of cell scrapers would be better?

Also, are the microglia isolated from this protocol in a resting state (M0), or are they in a activated state (M1 or M2)? If activated, do you think keeping them longer in culture would bring them to an resting state?

Thanks.

Best regards,

Rakesh

1)  Do you think using cell lifters, instead of cell scrapers would be better?

We have only experience with cell scrapers. I do not expect much difference but it is worth trying

2) Are the microglia isolated from this protocol in a resting state (M0), or are they in a activated state (M1 or M2)? If activated, do you think keeping them longer in culture would bring them to an resting state?

Hard question. They are somewhat activated, but not in what people call a typical M1 state. Keeping them longer in culture is not a good idea. In the absence of astrocytes microglia do not survive well. My advice is to use them 24h after isolation. I understand you would like to have a culture of isolated resting microglia, but I do not think a protocol to obtain this exists at this point. If you find it, let me know!

Best regards

Josep

I have also used this protocol to isolate microglia. I have found that when I serum-starve the microglia overnight (in serum-free DMEM) before LPS stimulation, they become more spindle-like and have small soma. They seem to be "resting" but I have not done further characterization on it. There's always the question of FCS in the medium that may activate the microglia. If anyone notice the same changes, please let me know!

Dear Dr. Josef,

Thank you for your advice. 

Trypsinizing the isolated microglial culture for 5min at 37degC, and then using the cell scraper works fine. But yes, you're right, there is a high mortality, but still I have some reattached microglia. A little more optimization should work better from next time.

@Ka Ka Ting: I too observe this. Overnight serum starvation of cells leads to round microglia cells, which is more 'resting' is suppose. 

Dear everybody!

I am also working with primary microglia according to the protocol of Saura et al. I have the same problem as Rakesh Sharma has. I have already tried to use cell scraper, but i haven't got living cells at all. However it was after an almost 4hours of trypsinization. Is it possible, that the reason was, because the cell walls were very weak after this long term trypsinization? I will try the 5mins at 37degC and then use the cell scraper. Thanks for answering.