Cd34 alexa 647 clone ram34

Satellite cells were isolated as described previously (32 (link)). Briefly, TA, gastrocnemius and quadriceps muscles of mice were subjected to enzymatic dissociation (0.2% Collagenase type II and 0.02 units/ml Dispase II; Sigma-Aldrich) for 30 min. Muscle was then minced and filtered through a 70-μm nylon filter and incubated with the following biotinylated antibodies: CD45 (clone 30–F11), CD11b (Cat. no. #553309), CD31 (Cat. no. #5011513), and Sca1 (clone E13–161.7) (all BD Bioscience and 1:150 dilution). Streptavidin beads (1:10 dilution; Miltenyi Biotech) were then added to the cells together with the following antibodies: integrin-α7–phycoerythrin (PE) (Cat. no. #R2F2; Ablab, 1:100 dilution) and CD34–Alexa 647 (clone RAM34; eBioscience, 1:50 dilution), after which magnetic depletion of biotin-positive cells was performed. The (CD45⁻CD11b⁻CD31⁻Sca1⁻) CD34⁺integrin-α7⁺ satellite cell population was then live cell sorted by flow cytometry using a FACSAria II Cell Sorter (BD Biosciences) with a 70-μm nozzle. Sorted cells were plated in DMEM, 20% FBS, and GlutaMAX for 48 h. Cells were then differentiated in DMEM and 2% horse serum for 72 h before being fixed for immunofluorescence.