Dear Melanie Vargas ,

There are some flaws in the suggestions above so I want to rectify them. Don't go for high-speed centrifugation for longer durations as that will compromise cell viability. PI should not be used in direct culture media as FBS, sometimes phenol red and many background contaminants can greatly influence the ultimate results. You can't go for PI incubation first and then trypsinizing your cells.

Please follow these steps, I hope it will clear your confusion and help to get what you are looking for-

-From your confluent wells, collect the cell supernatant in 15ml falcons (do not discard it). This will collect all dead cells which will be floating in cell culture.

-Now wash your cells with PBS once and trypsinize them.

-Collect these cells in the 15ml falcons in which you have collected respective supernatant.

-Centrifuge the cells, I will not suggest a high-speed centrifugation for longer durations (opt for 4000rpm for 5 minutes as it will settle down all cells without compromising viability).

-Wash the cell pellet with PBS once, to remove trypsin and excessive FBS (another centrifugation round).

-Incubate these cells with PI as per your standardised concentration for (10ug/ml should work fine)

-Incubate your cells for 15 minutes, wash them in PBS (to remove unbound PI as it can cause toxicity in cells and will penetrate inside healthy viable cells if incubated for longer durations) and acquire in FACS.

Hope it helps !

Hi

To test the toxicity of a drug/reagent, PI is not been considered as the best method. However, by theory PI protocol can be utilized to compare dead cells vs live cells. Well PI-staining for FACS is pretty straight forward. 1. Harvest cells and aliquot ~100000 cells/100 μL into FACS tubes. 2. Wash the cells twice with PBS buffer. 3. Resuspend cells in 100-500ul of FACS buffer. 4. Add PI (10g/ml) 5-25ul and mix well. 5. Incubate in ice for 1 min @ dark. 5. Determine PI fluorescence by FACS. Hope this will help you. 

My concern is to loose the dead cells if I centrifuge my cells, hence my question will PI still work if I add it directly to my cells ? I'm using FreeStyle Medium without FBS and antibiotic. Also 10g/ml seems a lot you mean 10ug/ml right ?

Thanks for your reply.

You are welcome. Oh Yes, it is 10 ug/ml. You can use a quick high speed sedimentation process (5-8 min @ 4000 g) to collect both the viable and dead cells or you could fix the cells after staining then your concern could be solved. PI surely works if you could add it directly to the cell culture. However by the time you process for FACS you get non-reproducible data due to false staining and or overstraining. We use PI for analyzing Necrosis, and use Annexin to score Apoptosis. All the very best.

Dear Melanie Vargas ,

There are some flaws in the suggestions above so I want to rectify them. Don't go for high-speed centrifugation for longer durations as that will compromise cell viability. PI should not be used in direct culture media as FBS, sometimes phenol red and many background contaminants can greatly influence the ultimate results. You can't go for PI incubation first and then trypsinizing your cells.

Please follow these steps, I hope it will clear your confusion and help to get what you are looking for-

-From your confluent wells, collect the cell supernatant in 15ml falcons (do not discard it). This will collect all dead cells which will be floating in cell culture.

-Now wash your cells with PBS once and trypsinize them.

-Collect these cells in the 15ml falcons in which you have collected respective supernatant.

-Centrifuge the cells, I will not suggest a high-speed centrifugation for longer durations (opt for 4000rpm for 5 minutes as it will settle down all cells without compromising viability).

-Wash the cell pellet with PBS once, to remove trypsin and excessive FBS (another centrifugation round).

-Incubate these cells with PI as per your standardised concentration for (10ug/ml should work fine)

-Incubate your cells for 15 minutes, wash them in PBS (to remove unbound PI as it can cause toxicity in cells and will penetrate inside healthy viable cells if incubated for longer durations) and acquire in FACS.

Hope it helps !

Dear Melanie and others

I am trying to do a similar experiment. I want to measure how many cells are dead in my adherent culture. So I followed the protocol of Ajaj pretty exactly. However, the population of dead cells in the FACS measurement is way too low than the actual amount of dead cells. I should have 30% dead cells, as seen under the microscope when adding PI directly to the culture. However, I only see 5 % in the FACS. My protocol is:

take off supernatant and transfer to eppi

wash cells in PBS and transfer PBS to eppi

trypsinize cells at 37degree for 3 min and transfer them to the eppi

spin down for 5min at 4degree, remove supernatant, add cold pbs, spin down again for 5 min, remove supernatant

then I add PBS containing PI to the pellet and pipette it up and down

The cells are then filtered and analysed in the FACS machine.

(I have done the centrifugation steps at 200g, 800g, 1600g, or 4000g, but was never able to get a decent amount of dead cells)

Can someone please help me?

Best

Clemens