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Annexin V assay analysis


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#1 Sayeh

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Posted 13 May 2010 - 03:00 PM

Hi everyone,
I have two basic questions. I am using Annexin V/PI assay to study the toxicity of certain drugs. I have two questions:

1) What determines the toxicity of a certain drug? Is it the percentage of the cells that are Annexin V positive and PI negative or the percentage of the cells that are Annexin V and PI positive?

2) In quadrant, we have four regions, UR, UL, LR and LL. There is plenty of explanation about UR, LR and LL regions but not on the UL region (PI positive and Annexin V negative). What do we learn from the cells in this region? If a cell is PI positive, wouldn’t it be necessarily Annexin V positive?

Thanks so much

#2 than4

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Posted 13 May 2010 - 04:36 PM

Annexin V is used to monitor apoptosis. It will only measure toxicity or death if your cells are dying by apoptosis. What parameter you choose to measure toxicity depends on whether you just want to know how many cells are dead, or if you want to know how many cells are apoptotic.

Cells that are Annexin V positive will have flipped their phosphotidyl serine and be in the early stages of apoptosis, but will not yet have their membrane integrity compromised and allow PI to enter and stain the DNA.

Cells that are both Annexin V positive and PI positive may be in the late stages of apoptosis or be necrotic. As the cell membrane is permeable you are unable to tell if the Annexin V that is stained is on the inside or the outside of the cell.
so, yes, cells that are PI positive should also stain for Annexin V as well.

For a toxicity assay, perhaps a more general cell death assay such as MTT/MTS or Trypan Blue/Cell Titre Blue might be more appropriate?

#3 Sayeh

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Posted 13 May 2010 - 06:34 PM

Thanks a lot for the reply. I am just confused as in some of my samples, I have about 70% necrotic cells (PI positive and Annexin V negative), and I don't understand how this can happen. As far as I understand, if cells are necrotic, they should be Annexin V positive as well. So I don't understand how is it possible to get PI positive, Annexin V negative (in UL part of the quadrant). I would really appreciate any help. I have seen papers using Annexin V assay for cytotoxicity studies but some of them use the UR (Annexin V positive, PI positive) as the percentage of apoptotic cells, some use LR and some use the sum of UR and LR as the percentage of apoptotic cells. Which one is correct?


Annexin V is used to monitor apoptosis. It will only measure toxicity or death if your cells are dying by apoptosis. What parameter you choose to measure toxicity depends on whether you just want to know how many cells are dead, or if you want to know how many cells are apoptotic.

Cells that are Annexin V positive will have flipped their phosphotidyl serine and be in the early stages of apoptosis, but will not yet have their membrane integrity compromised and allow PI to enter and stain the DNA.

Cells that are both Annexin V positive and PI positive may be in the late stages of apoptosis or be necrotic. As the cell membrane is permeable you are unable to tell if the Annexin V that is stained is on the inside or the outside of the cell.
so, yes, cells that are PI positive should also stain for Annexin V as well.

For a toxicity assay, perhaps a more general cell death assay such as MTT/MTS or Trypan Blue/Cell Titre Blue might be more appropriate?



#4 anokhi

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Posted 16 May 2010 - 09:28 AM

used to monitor apoptosis. It will only measure toxicity or death if your cells are dying by apoptosis. What parameter you choose to measure toxicity depends on whether you just want to know how many cells are dead, or if you want to know how many cells are apoptotic.

Cells that are Annexin V positive will have flipped their phosphotidyl serine and be in the early stages of apoptosis, but will not yet have their membrane integrity compromised and allow PI to enter and stain the DNA.
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#5 than4

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Posted 17 May 2010 - 05:49 PM

Thanks a lot for the reply. I am just confused as in some of my samples, I have about 70% necrotic cells (PI positive and Annexin V negative), and I don't understand how this can happen. As far as I understand, if cells are necrotic, they should be Annexin V positive as well. So I don't understand how is it possible to get PI positive, Annexin V negative (in UL part of the quadrant). I would really appreciate any help. I have seen papers using Annexin V assay for cytotoxicity studies but some of them use the UR (Annexin V positive, PI positive) as the percentage of apoptotic cells, some use LR and some use the sum of UR and LR as the percentage of apoptotic cells. Which one is correct?


I would agree that the cells you are seeing staining for PI only are necrotic, and am not sure why they are not also staining for annexin V.
Personally, I would not use the UR quadrant (Annexin V positive and PI positive) as a marker of apoptosis as you cannot distinguish between apoptosis and necrosis. I would use the LR (Annexin V postive, PI negative) or if doing a time course where you could see a "movement" from the LR to the UR quadrant, perhaps both.

As I've mentioned before, unless you are looking to see if your drugs induce apoptosis, I would switch to an alternative cell viability assay to establish toxicity.

#6 pesji

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Posted 17 December 2010 - 03:14 AM

Hi everyone,
I have two basic questions. I am using Annexin V/PI assay to study the toxicity of certain drugs. I have two questions:

1) What determines the toxicity of a certain drug? Is it the percentage of the cells that are Annexin V positive and PI negative or the percentage of the cells that are Annexin V and PI positive?

2) In quadrant, we have four regions, UR, UL, LR and LL. There is plenty of explanation about UR, LR and LL regions but not on the UL region (PI positive and Annexin V negative). What do we learn from the cells in this region? If a cell is PI positive, wouldn’t it be necessarily Annexin V positive?

Thanks so much

We have the same problem and was looking for explanations but couldn't find anything convincing. Cells might just get killed without going through apoptosis but if the cells are permable they should stain with Annexin V inside so I really wonder whether it's due to a bias of the concentration of Annexin V or any other explanation ???????????????

#7 Rsm

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Posted 17 December 2010 - 04:11 AM

Cellular debris (DNA) would be PI positive, Annexin V negative... Depends on your FSC/SSC gate, I guess.

rsm
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#8 pesji

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Posted 17 December 2010 - 04:20 AM

Cellular debris (DNA) would be PI positive, Annexin V negative... Depends on your FSC/SSC gate, I guess.

rsm

Cellular debris are excluded in the dot plot FSC/SSC and I only analyzed the R1 region events. In the meantime I had an extensive look on the net and found one possible explanation PI would enter even the living cells at a very slow rate and if the PI is added too long before analysis it would be seen as PI+/Annexin V -

Another possible problem could be the compensation between FL1 and FL3 (since I use Annexin V Fluos). Would you think that might create trouble ? I naively thought that FL1 and FL3 are too apart to need compensation ????????????

#9 Rsm

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Posted 17 December 2010 - 08:38 AM

Usually PI is in the PE channel (I always forget which FL that is, maybe 2?), which overlaps extensively with FITC (I am guessing that "FLUOS" is FITC, I am too busy to read through the product page). Of course you can use Per-CP Cy5.5 as detector, but that doesn't change the spill over to FL1. This is the reason why you should always use unstained - PI only - FITC only controls!

rsm
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#10 Denis Baev

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Posted 19 January 2011 - 07:29 AM

Cellular debris (DNA) would be PI positive, Annexin V negative... Depends on your FSC/SSC gate, I guess.

rsm

Not only debris but also necrotic cells

#11 Denis Baev

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Posted 19 January 2011 - 07:37 AM

Another possible problem could be the compensation between FL1 and FL3 (since I use Annexin V Fluos). Would you think that might create trouble ? I naively thought that FL1 and FL3 are too apart to need compensation ????????????

Replace PI by 7-ADD (ViaProbe) and be happy. This dye utilises FL-4 channel (far red), exitable by 488 and has no emission spectra overlap with FL-1.




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