Run-down of my protocol:
Dissect hippocampus into 500ul ice cold dissociation media (HBSS +sodium pyruvate, glucose, 10mM HEPES). Wash 2x with dissociation media.
Add 450 dissociation media + 50uL 2.5% trypsin, incubate at 37'C for 20 minutes.
Wash 2x with dissocation media.
Wash 2x with plating media (MEM Eagles + FBS, glucose, sodium pyruvate, glutamax, pen/strep).
Triturate tissue 8-10x with fire-polished glass pippette in plating media (here, I always make sure the tip opening is ~the size of a 1000uL pippette tip).
Count, plate, incubate.
When I count, I find that around 90% of my cells are staining positive with trypan blue. I've read up on a slew of hippocampal dissection articles, but can't find what I'm doing wrong. I don't know anyone at my institution who does this type of culture, or else I would ask them!
Thanks in advance!
You are doing everything right. But at some stage of the preparation there is probably a mistake. Please watch the film and the classic protocols to understand your mistake.
https://www.thermofisher.com/by/en/home/references/protocols/neurobiology/neurobiology-protocols/isolation-of-mouse-primary-neurons.html
Renee,
Your protocol looks fine to me as well. The diameter of the pipette tip is critical for getting a good balance of dissociation and survival. What if you try to go for a slightly larger diameter, just in case it's a little too small?
In my own cultures, I've switched from using the polished glass pipette to snipping about 3mm off of a sterile plastic P1000 tip (mostly because the neighboring lab that had the glass etcher moved :) ), and using that to triturate ~20 times until the majority of tissue chunks are gone. I find it's easier to be consistent, and can be done in a pinch. When processing big pieces of cortex instead of hippocampi, I dice the tissue with a razor before trypsin digestion, and triturate using P1000 tips cut to progressively smaller diameters (20 times with ~5mm diameter, 20 times with ~3mm diameter, 20 times with ~2mm diameter. Sorry, I should really actually measure these things!), then run the suspension through a cell strainer to filter out debris and remaining tissue.
Sara
Hi Renee,
I would also suggest using a larger opening tip for trituration step. I use sterile pasteur pipette for this. I also just prewarm the trypsin 15min prior to use and leave on the cells for 15min @ 37C before stopping the reaction with serum containing media (3 times the vol of trypsin). I then spin down at 1500 rpm for 3 min, aspirate the media & replace with media only - this is usually enough steps to remove the trypsin and can now dissociate easily with the pasteur pipette! I also agree to use the cell strainer to remove any debris or cell clumps that may not have dissociated fully (though this is usually negligible).
Thanks everyone for your answers ! I think I'll try trituration with a larger tip size first - that may be my problem.
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