I tried accutase and dispase but cells tend to differentiate. Probably something is wrong.
Usually I use the collagenase to maintain the culture undifferentiated because it is right to dissociate in small pieces and no in single cells. But now I'm going to infect this cells so I have need to dissociate into single cells to count and calculate the titer of infection and I know that it is possible, infact I have read some protocols for this aim but by my hand I have some difficulties. If somebody has some suggestion....thanks
Hi Clara, on a training in germany I were instructed to add ROCK inhibitor after doing single cell passaging of iPSC..hope helps
I have already tried, I used Rock at 10uM before and after the dissociation. Do you use this concentration or higher? ....Sorry an other detail... I am working in free feeders condition
For passaging we use TrypLE with soy bean inhibitor for inactivation
actually we used 2uM of Thiazovivin alternative is 10 uM Y27632 +bFGF 8ng/ml change to standard medium after 24 hours,
we also preconditioned the culture prior splitting with bFGF and ROCKi 2-4 hr before splitting
Fresh media+ 10nM Rock Inhibitor, 2+ hours;
accutase+10nM Rock Inhibitor，37°C，13 minutes;
Adding fresh media (3 times of the volume of accutase )+10nM Rock Inhibitor to the supernatant from last step;
Pipet 4-6 times;
Counting cell numbers;
Seeding+10nM Rock Inhibitor for the first 24hours.
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