Hello the community.
I had an interesting observation recently: I trypsinized HEK293T cells in a 12-well plate and afterwards, rather than adding complete medium, I added PBS to the cells. I transferred the cells to a tube and wanted to pellet the cells down by 500g for 5mins. However, the cells formed flocculant precipitates that floating in the PBS in the end and couldn't be pelleted.
I used a P1000 to forcefully resuspend the cells and used a viability dye to see if the cells were dead (as dead cells tend to be sticky), but they were >99% viable.
Does anyone know the reasons? Thank you very much!
Hi, @Jian Wang, after trypsinization the enzyme should be quickly inactivated by serpins present in the serum which is added to the complete medium. Otherwise the cells will be over digested and killed by the enzyme, which releases DNA and thus makes the cells viscous.
@Jian you should use Trypan blue dye instead of DAPI, the last one does not distinguish between live and dead cells, it stains DNA. Trypan blue will stain only dead cells.
@Jian, pardon, this is a really uncommon use of DAPI for me. Once I forgot to inhibit trypsin by the serum, the cells were not pelleted and were dead. So it is now a puzzle for me: why DAPI didn't stain the cells? As I understand you had at least 1% of the stained cells so DAPI was working?
Aitsana Maslakova That was not a problem at all and thanks for the replies. I was only correcting the statement and hope it could be helpful in the long run. It might be useful to have a look at DAPI product websites such as this one: https://www.thermofisher.com/sg/en/home/life-science/cell-analysis/fluorophores/dapi-stain.html#:~:text=DAPI%20is%20generally%20used%20to,popular%20cell%2Dpermeant%20nuclear%20counterstain. So basically it is the chemical property of DAPI that makes it less able to penetrate through live cells, whereas Hoechst 33342 dye can be used to stain DNA of both live cells and fixed/dead cells. Therefore, if DAPI is at a right concentration (between 0.1-10ug/mL), we can use it as a cell viability dye which selectively stains dead cells.
And yes, I totally agree excessive trypsinization will kill the cells, but it did not look like the cause of the problem in my case, since having 1% dead cells out of the whole cell population is usually not to be worried by my experience.
But nevertheless thanks for the advice.
I had something like that happen WITHOUT adding PBS, just trypsinization + spinning. The cells weren't dead either (though I used trypan blue not DAPI). I just assumed prolonged trypsinization causes that (I had to leave cells for like ~20 min because someone else was using the centrifuge :(( ).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line