Why there are more dead cells are present after one week of reviving?
I centrifuge the cells at low RPM and then wash with PBS and add fresh media to it but the no. of dead cells remain same?
Can anyone suggest me the reason for this?
Hello. The number of viable cells could be very low in the first place. There are few possible reasons for this. Maybe when the cells were transported to your institute, it was not in a proper dry ice that the cells died during shipping. It has to be quickly stored in liquid nitrogen immediately upon arrival, that if this step takes a long time, the cells may lose its viability. When reviving the cells, it is best to quickly unfreeze them. When adding fresh medium to the cells, add the medium drop by drop so that the cells will not get a shock from the change in medium environment. Lastly, it is best to leave the cells (in a fresh medium) on the bench (or in the incubator if you are worried about the medium buffer) for about 30 minutes so the cells can adapt to the new environment. With or without centrifugation (depending on what cells you are working with), upon transferring them to a T25 flask, try not to move the flask abruptly. The cells are trying their best to attach to the surface, so we do not want to disturb them. Hope that helps!
Amit,
Is this cell line one that is new to you? The reason I ask is that some cells will simply show a greater propensity for dying after reviving them than do other cells. Even if all things happen correctly while freezing and subsequently thawing the cells, many cell types will do quite poorly for perhaps the first four or five passages before they become "accustomed" to the culturing conditions. For instance, I have experienced that my cancer cell lines will begin growing quite well after only one or two passages, but my microvasculature cell lines will tend to have a significant number of dead cells in the flask for about two to two and a half weeks after reviving them, and this seems to be consistent with what others have observed as wells. In sum before you get worried that something is terribly wrong it may be best to ask a friend if this is normal.
Also does this happen to be a suspension cell line? If not then may I ask how frequently you are 1) changing the media, and 2) passaging the cells? These are just a few things that you may want to consider. Often times when cells are struggling after reviving, it is best to work with the cells minimally for a while, i.e. change media when you can get away with it and only passage when it is needed; these cells are in a precarious state and working with them tends to be hard on them. If they are suspension cells, then in general, it seems more difficult to get rid of dead cells as you cant just aspirate them off. Here it may be best to see if you can centrifuge the cells at an even lower speed and thus further reduce the accumulation of cell debris and or dead cells in the pellet.
I hope this helps,
Andrew
Thanks Andrew
I am working with DT40-cell line, which is growing in suspension culture. I am passaging 1 million cells in fresh 10 ml media and after exceeding to 10 million cells I am passaging the cells without centrifuging, to fresh media
Also, please suggest me the total no. of cells and viability required for transfection with electroporation purpose
Amit,
It is suggested that you culture your cells between 200,000 and 1,000,000 cells per mL and that you split these cells everyday given their doubling time of 11 hours. For transfection using electroporation, 20,000,000 cells per transfection tends to work well. If you have further questions, the following is a good resource for the cells you are working with: http://cwp.embo.org/pc10-17/docs/Sale.pdf
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