Hello!
I have been using Annexin V FITC Propidium Iodide from a commercial kit and got similar results twice. Unfortunately, for the third experiment, the PI of the kit got exhausted. Someone suggested to use PI in 100ug/ml conc. along with the kit Annexin and buffer. Since then my Annexin population, i.e. early and late apoptotic population is not visible (not even in positive control). Only PI positive cells, i.e. necrosis is visible. Can anyone kindly explain the reason?
Thank you.
Best!
it is always better to use kit components. but in your case you can try different concentration of PI along with annexin. most appropriately final PI concentration should be of 2 μg/mL in each sample.
Hello!
An Annexin V-FITC working concentration of 0.25 µg/mL
https://www.sigmaaldrich.com/catalog/product/sigma/apoaf?lang=en®ion=RU&gclid=EAIaIQobChMI75vqlrHr7AIVzOeyCh2n8AONEAAYBCAAEgIIB_D_BwE
Apparently you had a high concentration of PI, here is the link recommend 20-50 μg / ml PI. If the concentration of PI is high, then there may simply be light (the intensity of the signal from PI is greater) and only cells at the stage of late apoptosis and necrosis are visible, and not cells at the stage of early apoptosis, which annexin-5 detects.
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/propidium-iodide
Thank you Alexdanr. I will do it with the said concentration and revert..
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