Vcam 1 biotin clone 429

Muscles were dissected from mice and dissociated mechanically. All hindlimb muscles were used except in experiments where FAPs were isolated from VMOs injected into TA muscles. In this case, only the TA was dissected. The muscle suspension was digested using Collagenase II (760 U/ml; Worthington Biochemical Corporation) in Ham’s F10 (Invitrogen) with 10% horse serum (Invitrogen) for 90 minutes at 37°C with agitation. The suspension was then washed and digested in Collagenase II (152 U/ml; Worthington Biochemical Corporation) and dispase (2 U/ml; Invitrogen) for 30 minutes at 37°C with agitation. The resultant mononuclear cells were then stained with the following antibodies: VCAM-1-biotin (clone 429; BioLegend, 105704), CD31-APC (clone MEC 13.3; BioLegend, 102510), CD45-APC (clone 30-F11; BioLegend, 103112) and Sca-1-Pacific Blue (clone D7; BioLegend, 108120) at 1:75. Streptavidin-PE-Cy7 (BioLegend, 405206) at 1:75 was used to amplify the VCAM-1 signal. Fluorescence-activated cell sorting (FACS) was performed using BD-FACS Aria II and BD-FACS Aria III cell sorters equipped with 488 nm, 633 nm and 405 nm lasers. The cell sorters were carefully optimized for purity and viability and sorted cells were subjected to FACS analysis immediately post-sorting to confirm FAP purity.