Hi, I used for the first time the Annexin V-FITC Apoptosis Detection Kit (BioVision) but I am a bit unsure of the results.
I investigated the apoptosis on mussel M. edulis haemocytes, by using a Novocyte Advanteon Flow Cytometer Systems 1-3 Lasers, (Agilent), software NovoExpress 1.5.0.
The channel I used are FL1 (FITC-H) vs FL3 (PE-Cy5.5-H); by comparing with another study on mussels (not same specie) my opinion is that the group of cells of the right are apototic cells and the group on the left is supposed to be available cells. Still, I guess I am missing something because I double stained with Annexin and PI, but in my cytograms I dont see dead cells.
Can someone help me to clarify what Im seeing in these cytograms?
Fig. 1 and 2: untreated haemocytes (ctrl) 2x10^5 cells/ml
Fig. 3 and 4.: hameocytes exposed to microwave (to induce apoptosis) 2x10^5 cells/ml
Fig. 5 and 6: untreated haemocytes (ctrl) 5x10^5 cells/ml
Fig. 7 and 8.: hameocytes exposed to microwave (to induce apoptosis) 5x10^5 cells/ml
Thank you so much in advance!
Best,
Elisabetta
Maria Elisabetta Michelangeli Do scale your plots uniformly(x-axis and y-axis scale), you might get a better visualization of your experimental results then. Here it looks like each sample is gated differently. Usually, the gating settings for the control are applied uniformly to the treated samples to enable a comparison. Hope this helps.
It really seems like you do not have any dead cells but you can be sure by using POSITIVE (mortality) FMO and double stained controls. It meens that you take a small aliquot of your cells and simply leave them in RT for 3-4 days to die. After that, you stained them in three different ways: only with Annexin V FITC, only with PI and double stained them with both, Ann V and PI. Then the FMO and DS controls will make it easy for you to set proper gates and quadrants.
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