Anyone used Annexin V in their research?

[Awesome Sauceome]

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Hey anybody ever use Annexin V in their research before to determine apoptosis? I am struggling with an assay using that stuff and was wondering if anyone could maybe offer me some ideas on how to fix it.

 

[SplenoMegastar]

The buffers are generally the trick. What modality are you using to collect your data?

 

[Awesome Sauceome]

I am using a kit so I am not too worried about the buffers. I am using a 96 well plate reader. I am having an issue where my negative control cells have higher fluorescence (thus higher apoptosis) than my treated cells. Also, the harsher my treatments the lower the fluorescence - this is a trend that I have continued to see which is opposite of what I should be seeing.

 

[NickNaylor]

Is there any chance that whatever modality you're using to induce apoptosis is screwing up your dye?

 

[Awesome Sauceome]

Nah, it's definitely personal error here. My partner is able to do it fine with the same cells, equipment, etc.

It's something that I am doing but for the life of me I can't figure it out.

 

[NickNaylor]

Nah, it's definitely personal error here. My partner is able to do it fine with the same cells, equipment, etc.

It's something that I am doing but for the life of me I can't figure it out.

Well, whenever I have issues with protocols I go back to basics. Are you following the manufacturer provided protocol to the T (since it's a kit, I'm assuming they have a protocol for you to use)? Or are you using your own protocol? If the latter, perhaps have your partner walk you through an assay or watch him/her do it once and take detailed notes.

 

[Awesome Sauceome]

Following the kit completely

But I have tried probably a dozen variations as well. When I say I have legit tried everything, i mean everything.

It's probably stupid to do but I think I am basically going to shadow my partner while he does it, maybe it's something as stupid as just how he pipettes or something

 

[NickNaylor]

Not stupid at all, that's the way you do it. Banging your head against the wall trying to figure it out yourself is the worse option compared to just watching your partner.