I have done surface immunostaining with fixed yeast cell using polysaccharide specific antibody (AlexaFluor 488 conjugated). In Flowcytometry, both negative (unstained) and positive (stained) samples showed similar peak in FITC channel. what are the possible reasons? I have checked the stained cells under microscope where the signal was ok. but in microscope i had to increase the laser exposure a bit (0.9s). Is there any suggestion for staining or flow cytometry data acquisition?
Hi there, have you checked the unstained samples under the microscope? Did you only get a signal for the stained sample and not the unstained one at this exposure? Please provide pictures using the same imaging setup to enable us to assist you. Have you waited for the laser to warm up in the flow cytometer before acquiring the cells? Depending on the instrument, it can take up to 20-30 mins. Have you attempted to increase the PMTV on the flow cytometer and still got no signal? Have you used other antibodies or fluorophores successfully using the same flow cytometer/cells?
Hello,
Sending us an example of your peaks would be great. I you get only one peak, I think that your staining didn't work, all your cells are negative. Do you have a compensation control Alexafluor488 on compensation beads that shows both negative and positive peaks?
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