Hi, I saw that when I treated my cells with UNC0642 (histone methyltransferase inhibitor) and did apoptosis assay (AnnexinV/ 7AAD), then gated based on the control group and found that the whole population shifted above (like in the picture attached). In literature, this compound induced apoptosis. I tried different compounds and only UNC0642 had this problem. What can be the problem here? Has anyone experienced this shifting population in apoptosis?
Thank you!
Check the compensation parameters for 7AAD and Annexin V (BV421-A) voltage. Still, there are cells on right side Y axis in 4th quadrant, so check the input voltage for Annexin V . Its better to set the annexin Voltage keeping a positive control (like H2O2 treatment). Also, First quadrant shows live cells are with 7AAD-A stain, therefor washing and 7AAD concentration and protocol could be optimized based the cell types.
Dear Emily,
One possibility, it is that your UNC0642 experimental compound is changing the shape of the cells, making them slightly bigger. I will include H2O2-treated cells as positive control of apoptosis, to visualize the four subpopulations, after working with the compensation. Thus, you can set up the position of the quadrant with confidence. Best regards.
Dear Emily,
In addition, It should be convenient to decrease the voltage for both the FL1 and FL2 detectors. Thus, the events located in the right side vertical axis will be visible as indicated by Suman Sengupta. Also, these changes will help to locate the live/healthy cells (Annexin V-FITC and 7AAD negative) in the left bottom quadrant.
Best regards.
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