Apc conjugated anti mouse cd117 c kit

Phycoerythrin-conjugated anti-mouse Ter119 (Ter119-PE) (BD Pharmingen, 553673), APC-conjugated anti mouse CD117 (c-Kit) (BD Pharmingen, 553356), and FITC-conjugated anti-mouse CD71 (BD Pharmingen, 553266) were used for the surface labeling of cells. Purified anti-mouse CD16/32 antibody (eBioscience, 14-0161) was added to block nonspecific binding to FcγR. For each experiment, Sytox Blue nucleic acid stain (Life Technologies, S34857) was used to exclude dead cells. Flow cytometry was performed with a BD FACSCantoII or BD LSRFortessa, and data were analyzed with BD FACSDiva software.

BM cells from the femurs and tibias were collected by flushing with 20 ml PBS passed through a 25-gauge needle. After centrifugation at 1,300 rpm for 5 min, the supernatant was removed and the cells were then washed by ammonium chloride lysis. Cells were incubated first using a Lineage Cell Depletion Kit magnetic labeling system with the biotinylated lineage antibody cocktail (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], and Ter-119) for 10 min at 4℃ and anti-biotin MicroBeads (Milt-enyi Biotec) for an additional 20 min at 4℃. Positive immunoselection was performed with PE/Cy7-conjugated anti-Sca-1 (BD Pharmingen), APC-conjugated anti-mouse CD117 (c-Kit) (BD Pharmingen), SAV-PB, and a FACS Aria (BD Biosciences) using a flow cytometer.