I would like to perform detection of apoptosis with using Annexin V (detecting translocated phosphatidylserine) or Mitotracker Deep Red (stains mitochondria in live cells) in luteal cells of a cow by means of a flow cytometer. For technical reasons, I need to store cells for 1-2 days to perform tests on the cytometer. Therefore, I would ask whether there is a method of cell preservation/fixation and storage them for future cytometer assays ? Is it better to stain the cells before preservation or after just before the cytometer test ?
I have tried preserving the stained cells in dried ice, it worked but not for long time as I got different results for the same sample after few hours of preservation. For Annexin V exp. I usually add PI to detect the dead cells as well so it is preferable to stain the cells just before flow cytometer test because these reagents are toxic if left for long time.
Best
Hi Robert,
For Annexin V /PI staining it is recommended to measure the samples as soon as possible (we usually measure the samples within 30 minutes after staining). So for this staining fixation is not appropriate.
For other stainings we use 1-2% PFA in PBS to store the cells.
Please be aware that some fluorochromes are not stable and will not "survive" certain fixation methods.
Whenever we combine surface and intracellular stainings we use commercially available Fix+Perm kits.
Best
Wolfgang
Thank you for all the advices. I understand that using Annexin V on fixed cells could be problematic. Is there any different method for the determination of apoptosis which will be able to be applied on a flow cytometer instead of Annexin V, wherein the fixation of PFA cells is not a problem for stainig apoptotic cells. If I could to determine the mitochondrial potential with using Mitotracker or maybe there is different method. Maybe the determination of apoptosis in fixed cells is not possible to determine and I have to use non-cytometric methods.
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