Anti c kit

WT mice were lethally irradiated with 900 cGy TBI and transplanted with 5 × 10⁵ BM cells from donor mice. The survival rates of the mice were monitored for 3 months. Ten weeks after transplantation, bone marrow cells of the recipients were stained with anti-c-kit, anti-Sca-1, and anti-lineage cocktail antibodies (BioLegend, San Diego, CA, USA) to measure c-Kit⁺Sca-1⁺Lin⁻ (KSL) progenitor cells by a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA).

Total BM cells were collected from two femurs and two tibias and flushed gently to analyze the hematopoietic cells. Dissociated BM cells were then washed with 1% fetal bovine serum (FBS) in PBS. Cells were stained with any of the following antibodies for 20 min on ice: anti-CD16/32 (Invitrogen), lineage antibody cocktail-v450 (BD), anti-Sca-1 (Invitrogen, D7), anti-CD45 (BD, 30-F11), anti-CD45.1 (BD, A20 or Invitrogen, A20), anti-CD45.2 (Invitrogen, 104), anti-CD48 (Invitrogen, HM48-1), anti-CD150 (Invitrogen, 9D1 or BioLegend, TC15-12F12.2), anti-c-Kit (BioLegend, ACK2 or Invitrogen, ACK2), anti-CD34 (Invitrogen, RAM34), anti-CD41 (BioLegend, MWReg30), and anti-CD105 (BioLegend, MJ7/18). Goat anti-LepR-biotin polyclonal antibody (R&D Systems), Live/Dead dye (Invitrogen), anti-CD45 (BD, 30-F11), anti-Ter119 (Invitrogen, Ter119), anti-CD31 (BioLegend, MEC 13.3), and streptavidin (BD) were used to analyze the MSCs. Cells were then fixed and permeabilized using Cytofix/Cytoperm (BD) for 20 min during intracellular staining. Next, the cells were washed with Perm/Wash buffer (BD), incubated with rabbit anti-SCF polyclonal antibody (LSbio), and stained with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Invitrogen). Flow cytometry data were collected using LSR II (BD Biosciences), and the files were analyzed using FlowJo software (Tree Star).