Hi,
I am interested in staining cells in human whole blood by Flow Cytometry. As I have to use a CD-19 FC-IgG1 primary and then come up with anti-human IgG1secondary to detect the marker of interest, lots of unspecific binding might occur as human blood contains plenty of IgGs that can be detected by the secondary antibody. I was thinking of using the approach of pre-mixing CD19FC and anti-human secondary to form a complex prior to staining work? It might also be tricky as it requires specific ratio of primary to secondary and maybe a requirement of blocking/eliminating the excess unbound material?
is there a protocol I can follow for this matter?
Many thanks,
Mira
If you are following the protocol as mentioned below, and yet getting problem. Yoiu are suggested to make one tube having secondary antibody only, to be run before the tube having primary and secondary antibody. The fluorescence if Secondary alone with cell is to be subtracted from primary secondary both tube. Signal of Secondary alone is considered as non-specific and need to be nullified electronically.
If you are following the protocol as mentioned below, and yet getting problem. Yoiu are suggested to make one tube having secondary antibody only, to be run before the tube having primary and secondary antibody. The fluorescence if Secondary alone with cell is to be subtracted from primary secondary both tube. Signal of Secondary alone is considered as non-specific and need to be nullified electronically.
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