I have worked on a PC-12 cell line and I have a hard time transferring the cells. The problem is: it's very difficult to separate the cells into single cells after centrifuging, even if I mixed them well. Could you suggest me any specific method to do that? Thank you very much.
I agree with Peter, I've passed difficult cells through a needle to help prevent clumping...just be careful not to go too small with the needle as you may increase the amount of debris in your culture
Are you using Trypsin Versin? if not., its better you add versin to you Trypsin. Versin help u to separate the cells to single cells
I have tried both (Trypsin-EDTA 1x and scraper); It did not work well. Thanks for you suggestion.
Traditionally, cell clumps are dealt with via trituration, essentially pipetting them up and down several times. If you're doing that and it's not working, try a small gauge needle instead. Transfer the suspension into a sterile disposable syringe, cap with a 22 or smaller needle and gently expel the solution. Repeat a few times, as needed.
Also, if you're not already doing it, it may help to rinse the cells with calcium free buffer before the trypsin treatment.
I agree with Peter, I've passed difficult cells through a needle to help prevent clumping...just be careful not to go too small with the needle as you may increase the amount of debris in your culture
agree too w colleagues...I worked with PC12 cell..is the best way to obtain a single cell suspension...take care to do the procedure very slowly avoiding to produce foam!
We found trituration the best way to deal with PC12 cell clumps. We fire polish a glass pasteur pipette to adjust the diameter of the pipette opening. Triturate gently and slowly, avoid producing foam. Make sure to wet the glass pipette with media first to prevent the cells from sticking to the glass. You will end up with healthy single cells.
You could also pass your cell suspension through sterile nylon mesh or cell strainer, that way you will remove the biggest cell clumps without causing too much mechanical stress to your cells.
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