Hello,
I am performing flow cytometry on knockdown HT-22 cells. I have attached a picture for reference as to what kind of results I am getting. I have used the "Annexin V-FITC Apoptosis Detection Kit (with PI)" by Vazyme. I followed the exact protocol as indicated by the manufacturer.
I am performing this to analyze the detection and rate of apoptosis (if occurring) by the knockdown of the gene. My question is why are the cells being imaged in a diagonal line of sorts. Isn't it supposed to cluster based on living apoptotic and dead cells. If anyone can help me I will be grateful. Thank you
How do your unstained cells look like? Do you have appropriate controls like only Annexin and only PI for your cell line?
Md Wasim Khan Thank you for your reply. I figured out the problem. There was fluorescent leakage from one channel to the other. So I ran a compensation reaction first. Unstained cells and single dyes (only FITC and PI). There was a bit of autofluorescence in HT-22 cells which also caused issues. But once the compensation ratio was calculated the results were fine.
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