Troubles with labeling epithelial cells with CFDA-SE - Troubles with labeling epithelial cells with CFDA- (Apr/19/2005 )
Troubles with labeling epithelial cells with CFDA-SE
Hello,
I have some problems/unexpected results while trying to label adherent epithelial YAMC cells with SFDA-SE to assess (using FACS analysis) their proliferation characteristics in different conditions.
I tried two different protocols of the staining; in both of them I used final 10 µM concentration of the dye:
* The first means taking the cells off the plate with trypsine, washing them with HBS and re-suspension in HBS for 2X10(7)/cc before tagging them with (the same volume of the double-concentrated) CFDA-SE reagent,
* The second means tagging them directly in the plate while they remain adhere (without taking them off with trypsine) after washes with HBS.
Cells accept the dye wonderfully; first pick of fluorescence is reasonably high and narrow (good and even uptake of the dye by the cells in both of the protocols, with minimal fade in the following days); I was able to track this initial fluorescence peak for 7-8 days before it came to the magnitude that is about a twice as an auto-fluorescence intensity. But unfortunately I was not able to get subsequent picks. I controlled the proliferation by assessing the confluence of the cell growth, by counting the cells (using hemocytometer) and by WST1 colorimetric assay - and I know that at least 7-8 divisions have got place.
It seems to me that my cells are not synchronized (using light microscope one can easily see some of them divide and some not in every time point).
What could be a possible explanation?
Anybody?
Hello,
I have some problems/unexpected results while trying to label adherent epithelial YAMC cells with SFDA-SE to assess (using FACS analysis) their proliferation characteristics in different conditions.
I tried two different protocols of the staining; in both of them I used final 10 µM concentration of the dye:
* The first means taking the cells off the plate with trypsine, washing them with HBS and re-suspension in HBS for 2X10(7)/cc before tagging them with (the same volume of the double-concentrated) CFDA-SE reagent,
* The second means tagging them directly in the plate while they remain adhere (without taking them off with trypsine) after washes with HBS.
Cells accept the dye wonderfully; first pick of fluorescence is reasonably high and narrow (good and even uptake of the dye by the cells in both of the protocols, with minimal fade in the following days); I was able to track this initial fluorescence peak for 7-8 days before it came to the magnitude that is about a twice as an auto-fluorescence intensity. But unfortunately I was not able to get subsequent picks. I controlled the proliferation by assessing the confluence of the cell growth, by counting the cells (using hemocytometer) and by WST1 colorimetric assay - and I know that at least 7-8 divisions have got place.
It seems to me that my cells are not synchronized (using light microscope one can easily see some of them divide and some not in every time point).
What could be a possible explanation?