Currently, I am doing a research on NKG2D ligand expression on cancer cells.
I would like to use dual color staining, which are APC-conjugated Ab for ULBP-1 and PE-conjugated Ab for ulbp-2.
When I do it in single color, the ratio is 1:5 (ab:PBS), therefore I stick to this ratio when I prepare my antibody cocktail: 5ul APC-conjugated + 5ul PE-conjugated + 20ul PBS to one sample.
The results are weird when compared to single color. For instance, ulbp-2 expression has reduced in dual color setting compare to PE-conjugated anti-ULBP2 alone.
What is the problem?