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!
Hello Roberta,
a colleague of mine, Anne Wolf, is an expert on isolating brain microlgia and we did all sorts of experiments with the isolated cells, including pRT-PCR. However, we purified them with a MACS kit instead of FACS sorting. Perhaps you can contact her here on research gate. She will surely help. You can also have a look at our pupilcation in the Materials/Methods part for starters.
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All the best and stay healthy,
Marc
Hi Marc, thank you very much for your answer!
By the time I got your reply, my PI and I decided to go for magnetic CD11b separation, to avoid long hours at the FACS sorter, during which microglia may change their transcriptional signature.
Thanks again for directing me, I'll contact Anne for some help later on :)
Keep the cells cold (4C) to avoid the ex vivo activation states, as you are aware can be an issue. You may also consider adding transcription inhibitors as described here: https://www.biorxiv.org/content/10.1101/2020.12.03.408542v1
When checking for RNA quality be sure to not rely on nano drop. You will likely have too little RNA to measure accurately. Instead, try Agilent Bioanalyzer Pico kit. MACS should get you ~100K cells per adult brain. Younger animals will have better yield. This is a great resource regarding different extraction methods and potential yields: Article Isolation and Culture of Microglia
If you want to avoid the interphase layer you can certainly pellet to the bottom of the tube with no 70% percoll cushion; however, you may have some contaminating peripheral immune cells mixed in (which can usually be sorted out via FACS, but not through Cd11b MACS sorting). This may be minimal, but is a consideration for your downstream RNA analysis. Isolation is best done with a max of two brains pooled together. Once you isolate the microglia, more can be put together for the MACS sort. Pooling too many early in the extraction becomes problematic upon the initial dissociation and 100uM filtration-- your filter will get clogged. I personally use mechanical (not enzymatic) dissociation for microglia extraction as described here: https://pubmed.ncbi.nlm.nih.gov/30471926/
If you choose to use the dounce homogenizer, often less is more (over homogenization will kill your cells).
Good luck!
Hi Elliot J. Glotfelty , thank you for the very precious answer and for the details you shared!
Yes, tissues must be kept cold at all times, but the Percoll step limits this as reagents will be at RT.
About the gradients, although I transcardially perfuse my mice prior to brain isolation, I totally agree, I'll go for the tricky but safer and cleaner interphase band.
I like to process brains individually, especially with the Percoll, my question was more related to the downstream MACS separation, and you answered perfectly :)
I am also a fan of mechanical homogeneization for this type of experiment, but I was told enzymatic digestion for neural tissue is needed if I want to work with mRNA later on.
Thanks a lot Elliot!
One mouse cortex is enough for RT-qPCR. FACS for Cx3cr1-GFP mouse brain is easier to get pure microglia. You can use picoPure for RNA isolation in less than 100 cells of specific brain region. You can use RT-preAmp-qPCR to detect lower abundant transcripts.
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