I am looking for a protocol that would allow me to isolate live cells from mice adult hippocampus (7-8 weeks old). I am mostly interested on neurons and microglia.
At the moment I have tried this protocol:
1) Dissect 1 whole hippocampus in cold HBSS and chop it in few pieces (should I mince it with a scalpel?)
2) Transfer the hippocampus into Papain (20 U/ml) + Collagenase/Dispase (1 mg/ml) + 2% B27, HBSS solution and incubate @37C for 30min
3) Wash with DMEM-F12 media (enriched with 5% L-glutamine, 5% d-glucose, 5% Pen/Strep, + 2% B27)
4) Gently triturate tissue 10 times with a Pasteur pipette, 10 times with a 1000ul tip, 10 times with a 200ul tip in DMEM-F12 media (same as above).
5) Filter in a 40um cell strainer onto a cushion of 1ml DMEM-F12 media (same as above + 1% FCS) in a 50 mL conical tube and centrifuge at 300×g at room temperature for 5 min
6) Discard supernatant, resuspend in 1ml DMEM-F12 media (same as above + 1% FCS).
I then count the cells with a hemocytometer (cell dilution 1:10, no tryptan blue) but the number of alive cells that I can see through the microscope is pretty low, roughly 10%.
Any thoughts? Should I pull more hippocampi together?
Thanks,
Not sure but may be it will be helpful for you.
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Dear Matteo,
The protocol you found seems to along the lines of what I do as well, with some minor variations:
1) I used MEM (no red color, as I found that the pH indicator is somewhat harmful to fussy neurons) + Horse Serum (10%) + glucose + sodium pyruvate + HEPES + pen/strep + N2 supplement + glutamax
2) I used cold EBSS + HEPES (for pH buffer) --- as neurons sensitive and fussy you want to make them as happy as possible
3) I try to keep my dissections under 7 min per brain as I found it yields best neuronal survival. Also once you took one hippocampus out transfer it to a new petri dish (keep it on ice) with fresh EBSS+Hepes. Blood and other bits of tissue are detrimental to the neurons, so you want to keep your tissue as protected as possible. I use micro scissors to cut hippocampi into 3 pieces each ( https://www.wpiinc.com/surgical-instruments/scissors/spring-scissors/micro-scissors ). Do not mince the hippocampi as squishing the tissue damages cells, make sure the spring-scissors are sharp (I replace mine every 3-4 mnth when
4) You need to activate your papain prior to placing you hippocampi there (I do mix hippocampi from different animals all the time, usually 3-4 hippocampi per prep, never had a negative effect and I've been doing this for 11 years). To activate add papain to your dissection solution (EBSS + HEPES for me) and place into a water bath or an incubator (I use incubator) for 20 minutes. Once that time elapses I filter my papain to make sure everything is sterile through a 0.2um syringe tip filter
5) I place hippocampi into the activated papin from step 4 (above) and place the test tube (falcon 15 mL) into the incubator on it's side for 25 min. I do not use Collagenase/Dispase -- my advisor told me it was bad for neurons like 10 years ago and I stopped and I never had any issues, cells separate out very well.
6) After incubation I remove the solution carefully without touching the hippocampi bits and wash once with my culturing media (described in stem 1). The serum will deactivate the papain.
7) I add in fresh culturing media (I used 5mL per 3-4 hippocampi) and triturate carefully---the biggest thing here ---do not get any bubbles in, they are very bad for neurons. How many times I triturate really depends on the look of the cells--you want to break them down but not damage, and the fever times you triturate the better. I only use a P1000 and usually don't go more than 5-6 times.
8) I also don't use a filer because I never have visible clumps and the fewer manipulations the cells experience the better they do. Also limits chances for contamination.
Looking at your protocol I think you are under-digesting (your cells only get 10 min in activated papain) and over-triturating.
Please let me know if you have any Qs
I'll try to make some changes in accordance to your protocols and let you know whether I'll succeed
Thank you both Shahrukh Khanzada & Natalia Murataeva,