Microglia does change their morphology. However, your cells still look like mixed culture.

Hi, I also work on primary microglial cells from mixed glial cultures. I have never try to cryopreserve these cells. What do you mean with normal morphology ? If you want to talk about resting microglia (= inactivated microglia), the morphology corresponds to a ramified form (as your first image). However, after activation, these cells become amoeboid (the size of the cellular body increases and ramifications disappear). In your different images, amoeboid cells increase with time after anabiosis. Have you done viability experiments in these conditions? Your cells do not seem to be in a good health.

... moreover, have you done proliferation experiments (Ki67 staining) ? It will help you to control the health of your cultures.

This is addressed to Tony Heurtaux. Thanks for you advice.'Normal morphology' here means resting microglia. These cells respond effectively and rapidly to LPS, and also they have the ability to proliferate.In the third picture of my file, they look refringent and healthy under the microscope, is there any possibility that without stimuli,they turned into an undifferentiated state?

This is to Yi Quan. Thanks you for your answer.But how do you tell these are mixed culture ?

You can do the immunocytochemistry to test the purity. I do see other types of glia there.

I did Iba-1 staining,and they show100% positive staining,maybe there exsits false positive. So I will use a negative control next time. In my attached file,these cells all show Iba-1 positive. So I am wondering in my culture ,why do they look like that finally?

... microglial cells slowly proliferate in a resting state. Studiyng this proliferation parameter might help you. You can also use markers to determine if yours cells are in an undifferentiated state or not several days after anabiosis.

Concerning your photos, what are the different time points ? " In normal condition", if the medium is unchanged, microglial cells detach themselves from the bottom of the well and die (few days). Have you observed a decrease of the cellular viability? A simple trypan blue exclusion assay will be enough to answer this question.

It's quite strange because your 3rd image looks like BV-2 microglial cell line.

I think this is very strange too, I took the first picture at Day 1 after anabiosis ,then I changed with the fresh medium,and the second and the third pictures were taken at Day 2 and Day 3 respectively.

Was it a mixed glial culture-conditioned medium that you added to your cultures ? I say that because in normal conditions of culture (without an anabiosis step), microglial cells look like better in this type of conditioned medium contary to a DMEM/10% FBS/antibiotics medium.

It can also depend on the contents of media. Micriglia changes morphology even in same culture conditions in non-mixed cultures.

I think you have plenty of non-viable cells on that picture, do you coat the dishes with sthing?

..... Is it really primary microglial cells ?

You have the point Tony

I agree with Tony and also Yi Quan, in your fist image, I can see other type of cells (probably astrocytes) in your primary "microlglial" culture, and they seem to disapear over the time. Maybe this perception is just an artifact of the region where you took the image.

On the other hand, and on my experience, microglia don't like to be in fresh medium for a long time period, they rather prefer to stay in mixed glial condition media, otherwise, as you can see in your images, they start to turn reactive and die within 3-5 days...

Primary microlgia always adopt a ramified shape when are in resting-like state, however, in your images they seem to be reactive or proliferating... As Tony said... the refringent microglia and "rounded-shape" that you present in your images look like BV2 cells rather than primary microglia.

Thank you for your advice,guys. And if possible, would you please attach a picture of your microglia? I'm confused.

... I send you a photo of our primary microglial culture (Iba1 staining).

This is a pic of BV2 for comparison, and your third time point and these guys look very similar.

I would suggest that storing primary microglia is a bad idea. Glia are plastic, the very reason we study them, and epigenetic (and other unknown) changes that are bound to be induced by cropreservation is likely to alter your data.

I agree that there is a big fat astrocyte in your first pic and if you would like to prove purity, you will need to stain for Iba-1 and GFAP + DAPI. Astrocytes typically do not diminish in number - but as was mentioned, you may have just been unlucky to capture a random one in your pic.

For the media, we have kept primary cells in DMEM+10% FBS for up to 72 hours and they become slightly more 'BV2' like in appearance, but not anything like your third picture.

Good luck.

Yes, I agree with you completely. I realized it was a bad idea to storing these microglia.Thank you very much.

Dear all,

Thanks for a lovely discussion.

I would like to add: I have read, and experienced that the main morphology of microglial cells in-vitro is amoeboid type. Specifically, while obtaining the pure cultures from the mixed cultures, the no of typical ramified microglia seems to be relatively less. Plus, serum concentration does seem to affect the morphology towards amoeboid.

Though I have a question to ask too. I have noticed that without supplementing the microglial cultures with astroglial conditioned media, the cell proliferation and survival starts getting compromised right from the first day. Is it normal, for even with a conditioned media, we have observed that the cells do not seem very happy almost after 4 days or so..?

Also, is there any way to keep the microglial cultures healthy for extended periods of time?

Thank you in anticipation.

Sure that this topic is very interesting !!! Concerning morphology of microglial cells in vitro, it is surprising how theses cells can adopt different states in function of stimuli: pro-inflammatory state (amoeboid state) in presence of pro-inflammatory agents (LPS, cytokines, kemokines) or anti-inflammatory state (ramified state) in presence of anti-inflammatory agents (IL4, IL10). From my small experience, we have described that primary microglia from mixed cultures are cultured in mixed glial cultures-conditioned medium. In others media, microglia die rapidly. One of the parameter that can help you to say if yes or no your microglia cultures are healthy is to evaluate the morphology of your cells. In this way, one of my colleague has established that the better medium to keep them more longer is to do a ratio 1:1 with conditioned medium and fresh medium (DMEM +10% FBS + 1% antibiotics). However, as described by Poojashri Mishra, in all the different media that we have tested, the duration of culture of these "pure" primary microglia is not higher than 7-10 days.

Murine microglia are easily isolated from mixed brain cell cultures. It is ramified after a short resting state after a day at least. They never divide according to my best. I maintained them few days in HG-DME-1% FCS- 1% ITS medium and standard antibiotics.

Thanks again. It has been enlightening discussion.

Dear Yuan Lee, look into M.Stark et al. Histochemical J 24: 827-832 (1992) than you will see that you pictures show a lot astroglia (look to their endfeets).

G.W. Kreutzberg did a lot on microglia: see Microglia: a sensor for pathological events in the CNS Trends Neurosci 1996 Aug 19(8) 312-318. You will find his main publications in this article together with discussions on morphology, enzyme- and immunohistochemistry. Sincerely yours, Enrico Marani

Thank you so much,Enrico Marani. You have no idea how useful this is to me!

Dear Enrico,

Thanks a lot! It was really helpful!

This discussion has been really helpful to me. I am trying to isolate primary microglia to grow in culture (i only need them for ~48 hrs after isolation) and many of you in this discussion seem to have experience with it. There are magnetic and percoll techniques but I'm not sure which one is better. Is anyone willing to share a protocol that has successfully worked for them to isolate primary microglia to grow? I would really appreciate any advice or tips on this technique.

Magnetic.

check out the book http://www.amazon.com/Microglia-Methods-Protocols-Molecular-Biology/dp/1627035192

and

http://www.jove.com/video/50647/culturing-microglia-from-the-neonatal-and-adult-central-nervous-system

I do not think there is one best way to do it. It will depend on what you want to achieve with your culture and what markers you use if you go with magnetic approach (some may not work, others will; Percoll may give you more of a cell mix, but that may be a good thing, again depending on what you are looking to answer with the study). CD11b seems to be working with magnetic beads, but it is not an ideal marker, as it is too selective, unless you are looking for "activated" microglia (whatever that means to you).

I would like to isolate CD11b activated microglia to make conditioned media. My reading seemed to lead me down the magnetic isolation path but my lab doesn't have experience with it.

If you want Brandyn, you can have a look on our last paper on glial cultures

.... in this paper we have try to purify our primary astrocyte cultures. However, since several months (more than 1 year) we apply this magnetic protocol to perform primary microglial cultures. Before doing that, microglia were cultured after shaking our mixed glial cultures. It's clear that the output is better after a magnetic separation.

Shaking is simple and quite good procedure, even if it is an old one. However, the purity depends on last adherence process, and the homogenity surely not as good as by a modern method. But it is a cheap one - resulting mixed and slightly activated pool of cells.

You are totally right Attila. However, if you want lots of microglia, the magnetic separation is better.

As there is no good marker for a resting brain microglia, or they even divided for different subpopulations, an ab-based magnetic separation is a tough style of approach. but it can works surely, (i have no experience on it).

..... the CD11b microbeads antibody allows really a good positive-separation.

That beads will isolate macrophages freshly arrived from vessels too. Or blood-originated macrophages. This is an old story i think. Anyway, i used that marker to stain my microglia from mouse brain tissue.

Thank you Tony Heurtaux and Attila Lehotzky for your help. Attila if that is true is it standard to remove the blood by perfusing the mice before harvesting the brain? Or is there another technique to prevent macrophages in the blood from affecting the study? In your paper Tony it seems like you do not perfuse but remove any large blood vessels.

In this paper (in vitro data), we do not perfuse the brain. It's quite normal because, as you know, to perform primary glial cultures, we work on newborn mice. However, for in vivo experiments, it's crucial to perfuse animals before harvesting the brain.

Hi,guys! I think the paper"Physiology of Microglia" by Helmut Kettenmann is really helpful. Hope it can give you some hints too.

Thanks for all. valuable discussion.