Actually we are looking for the effect of some chemicals on fibroblast and in one of the treatment the newborn dermal fibroblasts are not getting detached from the plate. But in the same conditions in the control i.e. without treatment the cells got detached in two minutes. But in chemical treated condition they are not even changing their morphology at all after the same time of trypsinization. And after staining them with DAPI they are totally fine with prominent nuclei (not dead cells just attached to the plate).
Did you wash you cell before putting trypsin? If yes, could you change higher concentration of trypsin to detach the cells?
Hello Yonglian,
Yes I washed the cells before trypsinization . And even in control conditions cells got detached nicely but in the set of after chemical treatment they are not detaching.
3 suggetions:
1) Try higher concentration
2) keep longer time with trypsin ( for 7min)
3) impact the flask after reaction
1. How do you know they are not dead by DAPI staining? DAPI will stain DNA in dead cells just as well as in live cells, or better. You could check with trypan blue which will stain dead cells but not live cells.
2. If your treatment made the cells flatter or bigger or with stiffer cytoskeleton (but still alive), then the EDTA and trypsin may take longer to penetrate under the cells. Trypsin used for too long or at too high a concentration can damage cells (eg their growth-factor receptors), so one approach is to do an extra wash with EDTA-PBS alone before adding the trypsin. This takes the Ca and Mg concentrations lower and helps cells to round up. And it may also be helpful to use actually a lower concentration of trypsin, (like half normal, but keep EDTA the same by adding EDTA-PBS without trypsin 1:1) because this can be left longer on the cells without damaging them.
Hello Dorothy,
Yes I checked the cells by trypan blue staining also they are not getting stained. And in nuclear staining I saw some ells are showing the characters of apoptosis i.e. the some cells are showing nuclear blebbing.
- Badges
- Science method