I want to compare the cell cycle profile of an untreated sample to a chemotherapy-treated sample using several standard flow cytometry methods:
- PI + Click-it Edu + pHH3 (fixed)
- PI + Annexin V (not fixed)
1. Should my unstained control be obtained from the untreated or chemotherapy sample? Or, should each treatment have its own unstained control? If I have multiple doses of chemo treatment, should the unstained be obtained from the highest dose?
2. In the case of PI + Annexin V, where there are no antibodies involved, do I still need single stain controls? Measurement would be using 2 spatially separated lasers, so there will be no need for compensation.
3. If I am using 3 different lasers for the PI+ Edu + pHH3, do I still need single stain controls?
Just for clarity, how many tubes will I need to prepare for my experiment?
1. untreated - unstained
2. untreated - PI
3. untreated - Edu
4. untreated - pHH3
5. untreated - PI + Edu + pHH3
, then equally the same for the chemo treated = 10 tubes?
Here is what I did, after consulting another flow cytometry expert. Single stains and unstained controls should always be used.
Example experiment:
2 colors
5 treatment conditions
2 cell lines
Cell line 1: 5 treatment tubes with dual colors added, 2 single stain tubes using any of the treated cells (or a mix of treated cells), 1 unstained tube (similarly, any of the treated cells or mix).
Cell line 2: same as cell line 1.
Total = 8 tubes per cell line, 16 tubes total.
Ideally your cells won't have a difference in autofluorescence unless they are fixed differently. You should make a single stain control for all fluorescent channels used, including the dyes. The unstained controls can be from a negative population within your single stain control (usually better than unstained controls since there my be background staining with your antibodies/dyes). If you need a positive control for annexin V, you could heat-shock some cells, or leave them out on the bench overnight.
If you are only doing three colors, and they are on different lasers, you shouldn't run into many compensation issues (ideally), but it is always good practice to make your controls and apply the compensation matrix to your samples.
Also, for future experiments, double check that your live/dead dyes are compatible with fixation/permeabilization.
Good luck!
I just saw that you had added a response to your question..
Question 1. Should my unstained control be obtained from the untreated or chemotherapy sample? Or, should each treatment have its own unstained control?
That depends, if your treatment will result in different background/autofluorescence of your cells (which may be the case if it will have an effect on cell size/complexity). If your are working with cell lines, preparing one more control tube won't hurt.
If I have multiple doses of chemo treatment, should the unstained be obtained from the highest dose?
Pay great attention to whichever chemotherapeutic agent you are using in your in vitro experiments: many are fluorescent (all anthracyclines, ie. Doxorubicin, Daunorubicin, Idarubicin; Anthracenediones ie. Mitoxantrone; Chromomycins, etc.) and will stain your cells. Depending on the dose, time exposure and time to analysis they may be detected by FACS. This will also impact on the control you need to prepare.
2. In the case of PI + Annexin V, where there are no antibodies involved, do I still need single stain controls?
Yes, one single stain control for each fluorescence involved, regardless of it being a conjugated antibody or a chemical component. Spatial/temporal separation of lasers will not guarantee absence of spillover.
3. If I am using 3 different lasers for the PI+ Edu + pHH3, do I still need single stain controls?
Yes, bring all the control you can prepare. It is always best to have all possible control in FC, rather than discovering in front of the analyzer or - worse - during analysis that you should have brought one more single stain/FMO/etc..
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