You must gate all the cells that you want study. If you exclude the death ones, the result of PI will be biased.

For the incubation time tou must make a timing study. Each compound is different.

Thanks, Maria. I understand the optimization part and been doing it just wondering if someone had experience with pc3 cells.

Perform at time titration using your compound, usually over 24 hours. Indeed, you could perform an assay at fixed time points to assess the optimum dose/time. I have experience of PC3, this is what I would do as I do not know what your compound is. I would also gate the whole population on a negative control of healthy cells and look for early/late apoptosis based on the control cell culture flow cytometer morphology.

Thanks, Dan. I have been doing 24, 48 and 72 hours but looks like my compound is killing too much cells even at 24 h. My twice IC50 dose at the end of 24 hours has cell count only one third of the vehicle control which means I need to look at time points earlier than 24 h? Also, how does this correlates to biochemical events such as expression of Bcl-XL, DFF or AIF for that matter?

Thanks, Fernando. I am interested in both early and late apoptotic population so need to look at Annexin(FLH-1) and Annexin-PI (FLH-1+FLH-2) double positive. The confusion I have is gating at flowJo. I am selecting only the population is at the upper right corner (x-axis: FLH-2: PI; Y-axis: FLH-1: Annexin). Am I doing it right? I hope my made myself clear. Thank you, again.

You could get a mitochondrial assay too?

As for the high cell death, you can try on another cell line? Maybe dilute your compound or even have time points over hours instead of days?

To determine appropriate incubation time, 8, 16, 24 hr may be good starting point.

If you use attached cells, keep in mind that the floating cells in the media should be collected with attached cells.

Here are some references:

Clarke RG, et al. (2000) Cytometry 39:141-150.

van Gernderen H, et al. (2006) Nature Protocols 1:363.

Dan, Fernando and Yong:

Thank you guys for your suggestions. I have tried to tweak few things. I will let you know of the results in couple days.

Does your flow cytometer have a violet laser? If yes, consider throwing some Hoechst or DAPI in the mix. That way you can gate out debris and distinguish clumps. (Anything without Hoechst is debris, anything with more than the 2n amount of Hoechst is in S or M phase or is a clump.) If you do not have the Hoechst channel (violet leaser, blue emission), you can call molecular probes (now Invitrogen), they have a whole rainbow of (somewhat) cell permeant nuclear dyes.

Incubation times and concentration optimization have been already mentioned, also take the good advice on using untreated controls. If you have a lot of dead cells there, look at your cell dissociation protocol: the enzyme free dissociation buffers can do a serious number on the cells.

Good Luck!

Thank you, Anka. I am very new in flow so still understanding the basics. Its a BD FACSCalibur. At this point, I am only trying to make sure that I am gating the right population.

What was the cell count after 24 hrs when you treated with IC50 value? For determining IC50 value, how long did you treated your cells - 24, 48 or 72 hrs? You will obviously find less cell count if you treat with 2 IC50 value.

Gajendra, IC50 value was determined after 48 hours. I am loosing lot of cells ~50% for sure. Cell count reflected similar numbers. I am doing shorter time period this week. Let's see. Thanks.

Good luck. Update your result.

The value IC50 is usually determined at 48 hours after the agent application.

You want to use all the parameter at your disposal to select what appear to be single whole cells, so use scatter v PI to eliminate what you consider to be debris (probably low FSC/SSC, PI low to intermediate). Keep the gating consistent between samples.

less than 48hours is not ok

also if you have adherent cells resuspend them and wash them twice with PBS before staining

You should probably gate, but careful, since gating should avoid specific elimination of cells of interest. For instance, through your FSCvsSSC gate you could easily loose apoptotic cells. Compared to "live and happy" cells, apoptotics have lower FSC and higher SSC values, so be careful not to eliminate those ! Since annexin/apoptosis detection is a live cell assay, Hoechst or DAPI, as suggested above, will not help distinguish clumps or doublets from singlets, so, forget about that. You of course need a DNA stain to distinguish between necrotics and apoptotitcs (eg. PI). And you need to try to eliminate clumps/doublets through other means (area versus width). If PC3 cells are the only cell type you're interested in, then you're probably looking at a phenotypically (pretty) homogeneous situation, so, no gating other than on the indicated parameters.