How to dissociate N2A cells after centrifugation? - (May/05/2012 )
I've been culturing N2a cells for a while.
Generally, I use a PBS wash, wait a while, aspirate, wash with 5% Trypsin-EDTA, collect cells, place into a 15 mL conical tube, and centrifugate at 800 rpm for 5 mins. Afterward, I aspirate the solution off, place in new media, and attempt to dissociate the glob of cells at the bottom of the 15 mL conical tube.
That's where my problem is: Dissociation of the glob of cells at the bottom of the 15 mL conical tube after changing the medium.
How do I properly dissociate these cells?
I've tried using a 1 mL pipette, a 5 mL pipette, and 10 mL pipette. It seems like none of that really works. And it took a very long time to attempt to dissociate the cells with a 1 mL pipette (the long kind that goes into an electronic pipette thing).
I don't know how to properly dissociate these cells. How do I go about dissociating these cells so that they mix quite well in the N2a food media so there isn't an uneven amount of cells when I distribute portions of the cell collection to new flasks and/or for storage?
If the cells are clumping, it may be that you are over-trypsinising the cells. After adding trypsin, observe the cells on a microscope to see when they are detaching rather than waiting a defined time.
Depending on your centrifuge, 800 rpm may be too much and the cells are being sheared, releasing DNA and making the pellet sticky. You should be centrifuging at a maximum of 300 RCF (g forces), preferably 100-150 RCF.
Ok, let's assume i'm over trypsinizing them. Is there a way to bring things back into balance? Perhaps trypsin inhibitor? I sometimes use more than 1 mL of trypsin-EDTA to try and break apart cells. I do that, because I'm impatient, and because it seems like the process just isn't moving along.
I don't think 800 is too much. I've read other protocols, and 800 is the most I've seen. I could try lowering it to maybe 600 rpm for five minutes.
Adding more trypsin doesn't speed things up, it just means that you have more trypsin present. According to another post, these cells are quite firmly attached, so it could be that the clumps are a function of that.
Note that I was talking about RCF (relative centrifugal force) not rpm (revolutions per minute). RCF is a function of rpm and the rotor radius. RCF is the only way you can reliably talk about centrifuging as it is rotor independent, unlike rpm. 800 rpm MAY be lower than 300 RCF, but it may also not be!