I have a problem with the detection of a GPCR by flow cytometry.
Using a PE labelled antibody either directly or indirectly using a secondary antibody, I get a nice signal. Because we introduced RFP I need to switch to another channel. However, using the same directly labelled antibody either labelled with PE/Cy5 or Cy7 or using secondary antibodies labelled with these dyes we do not get any signal!
The cell line we use is several times shown to be positive and we can reproduce this as shown above. What is wrong? Does anybody have a solution? By the way, we are using a FACS Calibur.
It looks like the problem is in either antibody labeling or detecting Cy5 and Cy7. Have you been able to detect a signal from any other antibody labeled with the same dyes? If so - re-label your GPCR-specific antibody and try again. Sometimes antibodies loose antigen reactivity upon labeling so you may want to check if that is the case. Antigen recognision can be verified by western blot or ELISA.
Agree with Vasily. How is your PE control signals? PE control, Could be a cell or bead that is definitely binds to PE fluorochrome. If you receive ideal signals from PE control, the problem is related to your cells and antibody conjugation steps. But if you do not receive any signal from PE control, the problem is with your flow cytometry devise calibration or voltage setting. Wrong voltage setting is s common error in flow cytometry.
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