Hibernation media and genotyping via PCR to ease neuronal cell culturing

1c2bca1 Amy Palubinsky

If you’ve ever had to culture neurons from a single mouse embryonic brain because you don’t know its genotype, you probably think you know what hell is like. But there are ways to get around the many issues of individual culture—cross contamination, low yield, inconsistency between trituration across brains, maintaining sterility between animals, etc.—using a combination of tools and tips outlined here.

  • Hibernate Media from Brain Bits LLC: This amazing solution is a nutrient-packed medium that allows you to preserve the tissue you need in ambient carbon dioxide levels while you genotype your mice.
  • REDExtract-N-Amp Tissue PCR Kit from Sigma: This kit is for tissue PCR and contains everything you need to extract and amplify genomic DNA (from mouse tails, in this case) in roughly 20 minutes—yes, you read that correctly. There’s no mechanical disruption, organic extraction, column purification, or precipitation involved. After the last neutralization step, you’re ready for PCR using the kit’s Provided Reaction mix and your primers. And after you amplify, you can directly load the PCR product onto an agarose gel—no extra loading dyes, dilutions, etc.
  • Gel Red from Biotium: This is a nucleic acid dye that can replace ethidium bromide (EtBr) in your agarose gels without the lab having to change imagining systems or setups, as it has almost the exact same spectra as EtBr. That means no more special waste containers and having to call Environmental Health and Safety every time the trash needs to go out. And you don’t have to worry about those less-than-meticulous lab members touching things with their EtBr-covered gloves on.

So, how do you combine these tools to make that beautiful cell culture? Simple: While you're dissecting, take a tail sample from mouse No. 1 into one tube and place the tissue you need for culturing into another tube containing Hibernate Media and store at 4°C. Then repeat for mouse No. 2 through No. 1,000 (but hopefully less).

Once you’ve got your tail samples, you proceed to PCR using the super simple REDExtract protocol. Make your agarose gel with Gel Red in there instead of EtBr and let it set. When the PCR amplification is done, load the sample directly into the gel and run it out. Once you’ve got your genotypes, you can now pool all of the tissue from mice of the same genotype and continue with your dissociation and plating techniques. Yes, you’ve still got multiple tubes to deal with (depending on how many different genotypes you’re working with) but you’ve got pooled tissue, which means much less error during trituration and switching tools between groups. Your yield is most definitely improved, too, and you spend far less time in the seventh realm than you can imagine.

Note: Our PCR amplification takes almost four hours because we’ve got a big insert, so I actually amplify overnight, run the gel in the morning, and prepare the culture after the tissue has been in Hibernate for about 24 hours—and the cultures still look great. In the beginning, I did some troubleshooting and found that even after 72 hours in Hibernate at 4°C, I could still get beautiful cultures, so there seems to be ample wiggle room here.

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Amy M Palubinsky

Amy M Palubinsky

Amy Palubinsky is a Neuroscience PhD candidate at Vanderbilt University and mom to two human children and one fur-kid. She hates mornings and therefore loves coffee.