PI stain for cell viability~~Compensation? Suggestion needed~~^^ - (Apr/16/2012 )

Hello~~~

I'm new in FACS and I do have some question regarding about how to run FACS for PI staining and how to analyze the results.

1) If I only stained the cell with PI stain, do I have to do compensation? I only want to see the cell viability after treated with certain chemical compound. Usually I will do MTT assay but now my PI want me to do FACS for cell viability.

2) How i can interpret the data to see the effect of the chemical compound to the cell? I'm using HepG2 cells. Most of tutorials and reference that I've found onl related with DNA cycle, antibody etc. I'm confused, do I have to add FITC to my cell for PI stainng?

I really need some suggestions, please and thank you in advanced
~rpjkmust916~

1) no you don't need to do a compensation if you are only using one stain

2) wouldn't you just compare the number of dead cells in your treatment group to your control untreated group?

If you want to learn more about flow cytometry, Invitrogen have some excellent online tutorials:
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Analysis/Flow-Cytometry/Flow-Cytometry-Technical-Resources.html

Thanks Leelee,

I've watch the online tutorials and I have some question.

1) If the cells are dead,will I see red colour on the dot plot?
2) Should I used linear scale or log scale?
3) Where should I do the gating?

Again, thank uou in advanced.

~rpjkmust916~

In case you are still looking for answers

You don't need to add FITC. PI signal would come in the FITC channel.

1) No, You wont see a red colour on the dot plot. All the cells which are FITC+ would be PI+ and hence dead
2) Use a log scale
3) You can gate on the FSC-A vs SSC-A and look at everything. If you want to be more specific, you can gate out the debris by excluding the cells close to the 0 on the axes. Then look at the live cells and look for FITC staining.

Thanks~~~

rpjkmust916 on Tue Apr 17 01:58:29 2012 said:

Hello~~~

I'm new in FACS and I do have some question regarding about how to run FACS for PI staining and how to analyze the results.

1) If I only stained the cell with PI stain, do I have to do compensation? I only want to see the cell viability after treated with certain chemical compound. Usually I will do MTT assay but now my PI want me to do FACS for cell viability.

2) How i can interpret the data to see the effect of the chemical compound to the cell? I'm using HepG2 cells. Most of tutorials and reference that I've found onl related with DNA cycle, antibody etc. I'm confused, do I have to add FITC to my cell for PI stainng?

I really need some suggestions, please and thank you in advanced
~rpjkmust916~

Just running PI will not require compesation. But if you assess cell viability/ apoptotic cells or cells going into apoptosis: run also Annexin in an appropriate colour.