How Do You Detach Adherent Cells When Trypsin Is Ineffective?
In following up with research on what is trypsin/EDTA, I ran across the following, Trypsin:EDTA cell detachment process We always use PBS w/o Ca & Mg to wash our cell cultures prior to the addition of trypsin/EDTA. I remember one day many years ago one of the lab scientists just couldn’t get their cells to lift. Had about 8 flasks sitting in the incubator and the cells just wouldn’t budge. Turned out we’d received a shipment of PBS WITH Ca & Mg and nobody had noticed! You definitely need to use a Ca free buffer to wash your cells with when using trypsin/EDTA.
I would also try increasing the trypsin concentration. I use 0.1% trypsin/EDTA (I call it 2x) with primary umbilical cord MSCs and fibroblasts, and also with carcinoma cells lines in the past with much more reliable results. I’ve also sometimes had trouble with late passage cells or cells that had been growing without being subcultured for a while and the 2x trypsin helps. I find that adding a fair amount of trypsin (~2ml for 75cm2 flask or 100mm plate) and leaving it on also works better than removing the excess and leaving a “thin film” of trypsin. Also be sure to put the plates in the incubator in a single layer on the shelf rather than stacked. I’ve had MSCs that laughed at 0.05% trypsin, but came right off in a few minutes with the 0.1% trypsin. It might also be worth using a fresh bottle or aliquot of trypsin. Also, as csw alludes to, be sure you’re washing the plates well. If the cells are done and you’re just going to do something else with them (like get DNA/RNA, protein, etc.
Without knowing the specifics of your trypsinizing procedure, I think that csw has the right idea. I was taught to wash the cells with PBS without divalent ions after removing the medium from the cells but before trypsinizing and the only time I’ve had problems with getting cells to come off was when we accidentally ordered PBS with Ca2+ & Mg2+. I don’t, however, work with any really difficult cell lines (other than MEFs) but I’ve got coworkers and people in my department that do so I can ask around.
