Authors: Edwin M. Shen, Lisa V Salmeron, Gabriela Sanchez, Kara E. McCloskey
Flow cytometry, paired with fluorescent antibodies, is a popular method for characterizing cell phenotypes. Our laboratory is interested in deriving and characterizing vascular smooth muscle cells from embryonic and induced pluripotent stem cells, one of the few stem cell differentiation methods that remain underdeveloped. In our studies, we found that most commercially available antibodies advertised for smooth muscle cell identification using flow-activated cell scanning (FACS) were, in fact, not able to distinguish between positive and negative controls. Attempts to resolve the issues included exploring a range of incubation times, blocking reagents, staining kits, and titrating dilutions against both positive and negative control cells. In the end, we found that only the smooth muscle myosin heavy chain (SMMHC) antibody at a narrow titrating dilution range could distinctly bind to its intended epitope. Moreover, without more adequate and specific antibodies for labelling smooth muscle cells, we were not able to continue with our studies on smooth muscle cell fate.
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