Dear Reza,

                   The problem with adherent cell line is that while treating with trypsin-EDTA cells may die. To avoid this use non enzymatic reagents to dissociate the cells. That would keep membrane intact. You can use only EDTA solution. Other reagents are also available. Hopefully this will work for you.

Hi Reza,

you didn't mention how you detach the cells up to now. If you use a trypsin/EDTA based detachment you could alternatively try something like Versene, i.e. a purely EDTA-based agent without trypsin. Might be that your cells remain "healthier". In addition, or as an alternative, you might try to reduce the incubation time of your detachment procedure and better "loose" some cells which are still adherent but reduce the cellular stress.

(Have you ever tried to culture these cells in non-cell-culture pretreated dishes like e.g. those dishes for agar plates? Some usually adherent cell lines also grow well without the possibility to adhere.)

Best regards,

Markus

Hi Reza,

We do cell death measurements on adherent L929 cells by seeding them the day before in plates for suspension cells (non-tissue culture treated), as Markus suggested. They clump a bit together, so we resuspend them witn a 200µl pipette tip and pass them over a cell strainer to remove aggregated cells on the day of the experiment. Avoid to make bubbles during resuspension, some cells may go in (slow) apoptosis.

best regards,

sasker

I guess you always will have to consider some background of apoptotic cells, even with cells which are not so easy to push into apoptosis, like CT26, which prefer to undergo necro(pto)sis, e.g. after roentgen or particle irradiation (at least here on our site). Once you really manage to push your cells into apoptosis, you nevertheless will notice it even despite some background. As mentioned by Tamara, i also use accutase. 

Mowafaghyat baraye shomah wa khodahafez ! ;-)

Norman

Hi

For adherent CHO cell lines that I am using for FACS I use PBS with 2mM EDTA  which always seems to work well. I try to keep the cell in it for as little time as possible before washing and resuspending them in media.

Hi,

I usually use the LS174. These are cells that form aggregates. We used two protocols for  FACS that gave us little or no false positives :

1-Tryp +EDTA short time

2- Cell dissociation buffer

+ Filter 0.40

No problem.

Ice cold FACS buffer should work coupled with gentle scraping.  I have tried on Day5 Macrophages and it worked fine.   Annexin V should have some background when compared to the positive control.  I also recommend non-tissue culture plates.  Good luck. 

As long as you have the right controls, you would be able to tell if your cells are positively stained or not. You need a negative control Ab for background staining, which should be very close to unstained cells, usually isotype control Ab or non-relevant Ab. You also need a positive control for your staining, such as an Ab that you know is staining the cells. You also need a positive control for your relevant Ab of staining (the one that you are actually interested to know if it is staining your cells), such as cells that are reported to express the antigen for this Ab, or cells that are transiently or stably transfected with a plasmid coding for this antigen.

Dear Reza

Above comments are very useful, one thing I would like to add is "Apoptotic staining should not be done on cells with more than 80% confluency, because of contact inhibition the cells will start dying". Try doing assay at 50- 70 % confluence with proper negative and positive controls.

Best of luck

Dear Reza

I dont think that the problem is with the dettachment agent or confluence. Because even at higher confluence cells stop division but they tend to be live untill you keep on changing your culture medium regularly (this is the scenerio in most of stem cells). In case of cancer cells, some cells are already in a process of apoptosis so you can get a 8-10% apoptosis in cancer cells at control level. What you require to avoid false positive in case of a FACS protocol is gentle handling of your cells. While trpsinizing (doesn't matter you use trpsin-EDTA, only EDTA or accutase) pipette your cells manually as soon as there is a distortion in morphology and that is acheived within 2 minutes in case of epithelial cancer cells if you do gentle shaking at intervals while incubating at 370C, next is centrifugation step at 2000-3000rpm for 3-5 minutes. A very gentle retropipetting and a gentle resuspension of cells while doing washing step and last is the volume of annexin you add which depends on kit but keep in mind that you use exact number of cells for that staining volume and not less. My one colleague encountered the same problem and these suggestions worked very well there and we do obtain good reults..Hope it helps..!!

Dear All

The test was repeated with tenfold dilution of trypsin-EDTA (final concentration 0.025). The test process is done in cold condition. But I still have high false positive rate (80 percent).

What can I do now?

Dear Nathalie M. Mazure

what is your formulation  of Cell dissociation buffer?