# FACS spinal cord microglia **URL:** \ **Category:** Experimental Techniques\ **Created:** [February 21, 2019, 5:41am UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560 "2019-02-21T05:41:41Z")\ **Posts on this page:** 9\ **Page:** 1
**Author:** ![ShanTan](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/shantan/32/242_2.png) [@ShanTan](https://forum.painresearcher.net/u/ShanTan)\ **Post date:** [February 21, 2019, 5:41am UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/1 "2019-02-21T05:41:41Z")
Hi all, Wondering if anyone has had experience isolating microglia from spinal cord tissue? I’ve tried various protocols (Dounce homogenization and enzymatic digestion) with relatively low yield. Looking to do RNA seq afterwards and am having too low of a yield to do so. I gate with CD45 mid/CD11b+/CX3CR1. Any help on this is greatly appreciated! Of course these protocols works beautifully in brain, way less successful in spinal cord. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [February 21, 2019, 6:30pm UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/2 "2019-02-21T18:30:21Z")
@shantan I’ll preface this by saying I’m not a microglia expert, but I work with spinal cord dissociation quite a bit. Hopefully some microglia experts like @esypek can chime in. Sounds like you’ve been following the standard protocols. Spinal cord is definitely harder in most ways compared to brain, largely, IMO, because the large amount of axon and myelin. Dissociating neurons from spinal cord is very difficult too. This is why I use nuclei for nearly all my genomics work where I need to get some cellular RNA out. Although there are reasons to not do that as well. I’m sure you’ve seen this paper, but in case you haven’t: > **[Persistent Alterations in Microglial Enhancers in a Model of Chronic Pain -...](https://pubmed.ncbi.nlm.nih.gov/27184839/)** > > Chronic pain is a common and devastating condition that induces well-characterized changes in neurons and microglia. One major unanswered question is why these changes should persist long after the precipitating injury has healed. Here, we suggest... And here, Haring et al. show an optimized protocol for spinal cord dissociation with the targeting of neuronal cells, but likely will help for microglia: > [@Neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types | Nature Neuroscience](https://forum.painresearcher.net/t/neuronal-atlas-of-the-dorsal-horn-defines-its-architecture-and-links-sensory-input-to-transcriptional-cell-types-nature-neuroscience/428): > > [https://www.nature.com/articles/s41593-018-0141-1](https://www.nature.com/articles/s41593-018-0141-1) Marques et al. isolated oligodendrocytes from spinal cord for single cell: > **[Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous...](https://pubmed.ncbi.nlm.nih.gov/27284195/)** > > Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult... Some questions: 1. How many microglia do you need? You say > Looking to do RNA seq afterwards and am having too low of a yield to do so What yield do you think you need? The RNA-seq library prep will determine this. And in my mind, too low is no longer an issue. If we can do RNA-seq from a single cell, we can do anything. I routinely do sub-nanogram levels of RNA using the Smart-seq2 protocol or the Nugen SoLo ([https://www.nugen.com/products/ovation-solo-rna-seq-system](https://www.nugen.com/products/ovation-solo-rna-seq-system)). If you want to do amplification-free, or you’re trying to pull down ribosomal mRNA, that’s a different story. You need to increase your yield, which means more cells. But if you’re just trying to do regular gene expression profiling, the low-input methods I mention here will take whatever you give them (down to a single cell). Now, if you want to try to increase cell yield, there are a number of ways: - Pool animals - Optimize your dissociation protocol to (1) increase viability (2) increase number of cells liberated from the solid tissue, or ideally both. In your protocol you’re using, it could be that you’re freeing up a good number of microglia but they’re not surviving (and thus, viability is the issue). In that case, you want to do things that help with that (oxygenating your buffers, working fast, using the gentlest protease, douncing more gently). Do you check your preps at each step to get a sense of where our losses are? What number of viable cells do you get? These questions lead me to the next question/recommendation. Are you willing or able to use a transgenic line such as Cx3cr1-GFP or Sall1-GFP? That would allow you to remove the antibody labeling steps, which will likely increase viability (reduce time to FACS) and make your life easier. Also, because they’re fluorescent from the get-go, you can optimize your protocol better because you can look under the scope at each manipulation you do. The FACS sorting will likely be cleaner too since fluorescent proteins don’t have the background binding issues that antibodies do. If you’re not against using a transgenic, I recommend this. Note, however, that some researchers don’t like the transgenic lines because they believe that the physiology of the microglia are affected by the transgenes. This is particularly true of Cx3cr1-GFP, which is a knockin. So effectively you’re working with a heterozygote. Alternatives are a Cx3cr1-GFP BAC. Note sure if there are others. Here is the Sall1 mouse: > **[Sall1 is a transcriptional regulator defining microglia identity and function...](https://pubmed.ncbi.nlm.nih.gov/27776109/)** > > Microglia are the resident macrophages of the central nervous system (CNS). Gene expression profiling has identified Sall1, which encodes a transcriptional regulator, as a microglial signature gene. We found that Sall1 was expressed by microglia but... Lastly, if you’re not committed to using cells, you can use nuclei, in which case you’ll still need a transgenic to label the nuclei. Benefits of nuclei are that the dissociation is easier and likely less biased in terms of cell type capture since you’re not having to keep cell membranes intact. You just dounce and go. You can use frozen tissue too. I use that strategy here to get SST+ neuronal nuclei from the DH: > **[Transcriptional Profiling of Somatostatin Interneurons in the Spinal Dorsal Horn](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931607/)** > > The spinal dorsal horn (SDH) is comprised of distinct neuronal populations that process different somatosensory modalities. Somatostatin (SST)-expressing interneurons in the SDH have been implicated specifically in mediating mechanical pain.... One additional consideration. If you’re going to isolate live microglia and look at their transcriptomes, your dissociation is going to induce some gene expression just from the prep. This is an important issue for everyone doing single-cell or bulk RNA-seq on dissociated cells. We cannot ignore it. Discussed here: > **[Molecular Diversity and Specializations among the Cells of the Adult Mouse...](https://pubmed.ncbi.nlm.nih.gov/30096299/)** > > The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in 690,000 individual cells sampled from 9 regions of the... > **[Europe PMC](https://europepmc.org/article/PMC/5665481)** > > Europe PMC is an archive of life sciences journal literature. > **[Single-cell sequencing reveals dissociation-induced gene expression in tissue...](https://pubmed.ncbi.nlm.nih.gov/28960196/)** > > Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations If your question for RNA-seq revolves around seeing genes induced by injury or inflammation, you’d do best to reduce this dissociation-related gene induction as best you can. The method used here in Hvratin 2018 would be advisable (this goes for everyone doing cell dissociations, IMO): > **[Single-cell analysis of experience-dependent transcriptomic states in the...](https://pubmed.ncbi.nlm.nih.gov/29230054/)** > > Activity-dependent transcriptional responses shape cortical function. However, a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and... So summary: - Decide what “too low” is for RNA-seq. If this is your main concern, it might not even really be a barrier depending on the library prep you use - Consider using transgenic microglia lines - Systematically optimize your protocol based on the published ones, paying attention to artefactual gene expression - Consider nuclei if you’re not committed to cells @tberta @liz ---
**Author:** ![ShanTan](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/shantan/32/242_2.png) [@ShanTan](https://forum.painresearcher.net/u/ShanTan)\ **Post date:** [March 21, 2019, 4:06pm UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/3 "2019-03-21T16:06:03Z")
Hi everyone, just wanted to follow up on this. I’ve done quite a bit of tweaking of various protocols the last few weeks, and I have a protocol that works beautifully. I am getting around 50,000 microglial cells with a pooling of 4 lumbar spinal cords. More than happy to share if anyone needs it! The biggest thing is bubbling your solutions and keeping everything cold. I use a combo of mechanical and enzymatic digestion, unfortunately can’t seem to avoid enzymatic digestion which I know can be problematic. Mechanical alone does not do the trick. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [March 21, 2019, 4:54pm UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/4 "2019-03-21T16:54:38Z")
That’s great @ShanTan! Thanks for coming back to share the update and for being willing to share your protocol. Good luck with your ongoing experiments! ---
**Author:** ![RitaSilva](https://avatars.discourse-cdn.com/v4/letter/r/9de053/32.png) [@RitaSilva](https://forum.painresearcher.net/u/RitaSilva)\ **Post date:** [November 23, 2020, 9:26pm UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/5 "2020-11-23T21:26:01Z")
Hi Shan - some of my colleagues have tried to isolate microglia from the spinal cord but got a very low yield… would you be happy to share some tips? Thanks a lot in advance, Rita ---
**Author:** ![ShanTan](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/shantan/32/242_2.png) [@ShanTan](https://forum.painresearcher.net/u/ShanTan)\ **Post date:** [November 25, 2020, 1:40am UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/6 "2020-11-25T01:40:01Z")
Hi Rita, Yes of course. It’s not an easy prep and there was certainly lots of variability in the number of cells we would get during optimization. If you send me an email shannon.tansley@mail.mcgill.ca, I can send you our protocol. Cheers! ---
**Author:** ![RitaSilva](https://avatars.discourse-cdn.com/v4/letter/r/9de053/32.png) [@RitaSilva](https://forum.painresearcher.net/u/RitaSilva)\ **Post date:** [December 1, 2020, 7:27am UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/7 "2020-12-01T07:27:14Z")
Hi Shan, Thank you very much!! I’ve sent an email recently - have you received it? Thank you!! Rita ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [December 4, 2020, 3:04pm UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/8 "2020-12-04T15:04:46Z")
Hey all, You may be interested in this hot-off-the-press preprint on effects of different isolation methods on microglia > **[Single Cell Sequencing Reveals Glial Specific Responses to Tissue Processing...](https://www.biorxiv.org/content/10.1101/2020.12.03.408542v1)** > > A key aspect of nearly all single cell experiments is the necessity to dissociate intact tissues into single cell suspensions for processing. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that... @tberta @esypek @thicunha ---
**Author:** ![thicunha](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/thicunha/32/386_2.png) [@thicunha](https://forum.painresearcher.net/u/thicunha)\ **Post date:** [December 4, 2020, 5:50pm UTC](https://forum.painresearcher.net/t/facs-spinal-cord-microglia/560/9 "2020-12-04T17:50:35Z")
Great @achamess. We are going to run our first scRNA seq in two week and it will be helpfull.