Monocytes die after cell scraping - (Oct/17/2011 )
I am having trouble with viability following scraping monocytes. Briefly, I thaw human PBMC in warm RPMI, spin, resuspend in warm RPMI, plate in 6 well plates and incubate 1-2hrs at 37 degrees. I wash off nonadherent cells with warm RPMI, add 1 ml warm RPMI per well, check cell layer (adherent cells), then scrape cells and collect into conical for counting. However, when I count cells in trypsin, all appear dead (ie. all cells are blue). Could this be an artifact of the scrapping? Or does the scrapping in fact cause the cell death?
Thanks in advance for any advice/suggestions!
Scraping is pretty harsh on the cells. Basically it rips the cells off the surface they are attached to. If you can't use trypsin, perhaps try lifting the cells with a dilute solution (0.5 mM) of EDTA in PBS.
As usual Bob1 is correct...scraping the cells is the worst way of collecting cells. There are many alternatives to using Trypsin which again can be the harshest enzyme to use.
Pronase, Hyaluronidase, collagenase, Elastase etc
All can be used and one of the experimental optimisation steps for collecting your Monocytes would be to test each enzyme (including Trypsin) at DIFFERENT CONCENTRATIONS and TIMES OF EXPOSURE. You could also include different TEMPERATURES as well.
Hope this gives you something to go on.
P.S. Another variable I have just thought of is the TC plastic.......you can test out different TC manufacturers....what you are after is the adherence of Monocytes after 2 hours BUT have them very loosely adherent i.e. to make them as easy as possible to collect....only another thought.
"If experimentation was easy then everyone would do it" Uncle Rhombus.