I trypsin cells to do cell counting and seed in 96 well plate. Since I am a beginner, it takes so much time. Can I store the tube with cells in the incubator till I finish counting and then take the cells out for dilution and reculture the rest in a flask?.
And if you need after to add a certain volume to your pellet of cells (counted cells), you can do second centrifuge , eliminate the medium containing serum (that bloc trypsin action), and then adjust your X volume ... (The cells have not to stay longer with trypsin, The action of tryspin is just to detach the cells and then you have to stop it action )
Good luck !
I agree with Rodolphe that the cells would be safe after deactivating trypsin with serum containing media. I normally rinse the flask with media after trypsinization, centrifuge, aspirate all the trypsin/media mixture out, and resuspend in fresh media. I've used this method with human primary mesenchymal stem cells and the control wells had expected viability and proliferation rates despite an hour long seeding marathon.
Thanks all. I do add medium to stop trypsin. After, it takes much time to count cells. Can I store the tube during the time of counting? I don't know how long cells should be out of incubator.
It's best to minimize time spent outside of the incubator since they are very sensitive to any change in environment. If you're looking at protein expression for example, you may have an issue with traditional techniques. If you're doing an MTT where you're just seeing if cells survived, you should be fine leaving them out for maybe hours?
Since there is relatively little surface area when in a centrifuge tube, the incubator atmosphere will be almost irrelevant so it's mainly temperature you're worried about. Considering we can freeze these cells and some even put them on ice, this should be a small source of error considering all the other ways we torture the cells with pipetting and digesting. Keep good aseptic technique and do things to the best of your ability since truthfully, nobody knows all the effects in vitro techniques will have on cells.
Hi Heba,
What I used to do after blocking trypsin with medium (specifically, FBS in the medium), is to centrifuge again, decant the medium and resuspend the cells in 1 ml fresh medium, and adjust their dilution after counting. It is important in some cell types to not leave them pelleted after centrifugation for long time.
I used to leave it in the laminar flow. If your cells are temperature sensitive you could put them in the incubator if you worry about metabolites and gene expression, you should put them on ice.
- Badges
- Science method