Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

PI stain for cell viability~~Compensation? Suggestion needed~~^^


  • Please log in to reply
5 replies to this topic

#1 rpjkmust916

rpjkmust916

    member

  • Active Members
  • Pip
  • 25 posts
0
Neutral

Posted 16 April 2012 - 05:58 PM

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 Posted Image
~rpjkmust916~

#2 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
52
Excellent

Posted 16 April 2012 - 10:26 PM

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.invitroge...-Resources.html

#3 rpjkmust916

rpjkmust916

    member

  • Active Members
  • Pip
  • 25 posts
0
Neutral

Posted 16 April 2012 - 11:47 PM

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~

#4 zodiac1505

zodiac1505

    Enthusiast

  • Moderators
  • PipPipPipPipPip
  • 92 posts
3
Neutral

Posted 04 July 2012 - 11:15 AM

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.

#5 rpjkmust916

rpjkmust916

    member

  • Active Members
  • Pip
  • 25 posts
0
Neutral

Posted 30 April 2013 - 07:18 AM

Thanks~~~Posted Image

#6 knuko

knuko

    member

  • Members
  • Pip
  • 2 posts
1
Neutral

Posted 12 November 2013 - 12:13 PM

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 biggrin.png
~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.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.