I am stuck with the dilemma of my SH SY5Y cells looking like they are clumping and growing on top of one another with some space around them. They are not spreading around very evenly like some of my colleagues described. Funny enough, one colleague of mine is using 0.2% trypsin, another using tryple and another using accutase all subjecting to SH SY5Y cells and they hardly have any issues like mine.
Note that I have tried about 10 rounds of splitting from a revived batch from 10 separate cyrotubes and most of them ended up curling up like a ball and float - which I believe signifies cell death.
I have tried various concentrations like 2 mM EDTA, 0.02% Trypsin in 2 mM EDTA, 0.2% trypsin in 2 mM EDTA, 0.2% trypsin in 0.53 mM EDTA (this one following ATCC protocol) and even tryple and they still show signs of clumping after that.
These are the questions that I need answering:
1. Should I try accutase as I read its similar to EDTA but much less harmful from the rest?
2. The only suspected issue left is that after lifting the cells from the T25 flask, and then centrifuging them, I did not do a thorough rinse of the T25 flask with either PBS or with media in FBS to either further neutralize the trypsin/EDTA/tryple or remove any left overs of those cell adhering solution. Is that the only problem that I have?
3. Before trying out different cell adhering solutions, I used to use 2 mM EDTA alone and I used flask knocking to adhere the cells before centrifuging after subjecting it for 5 minutes. On the other hand, many have told me that when they were using their choice of the cell adhering solution, they wait until the cell lifts up and becomes spherical and they didn't need to knock. Should I follow suit and exposed the SH-SY5Y cells longer to EDTA until it becomes spherical?
4. Among the solutions listed in the title, which is more harsh to least harsh that they would reduce the chances to lyse the membranes of the cells when exposed too long? And do you recommend other types of "cell lifting" solutions other than the ones listed above?
Would be great to get your feedback as I understand from many of the different researchers that ppl used different cell adherent solutions with different timings and still get the result they needed. Thanks
Quick question....Do you typically wash the cells with DPBS before adding the trypsin? The proteins in the media bind to the trypsin/neutralize it and you don't get proper single-cell suspension formation when replating. For very stubborn adherent lines, I would wash twice with PBS, aspirate, and then add trypsin. 0.2% with EDTA should do it.
0.2% with 2mM EDTA (2 ml working sol in a T25).
1-2min in humid CO2 incubator (37C).
No mechanical jerk or tap, just add 10 ml complete media and wash cells gently out.
all the best.
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