Cd150 bv650

Adult, 8- to 15-week-old mice were injected intravenously with 0.8 mg/kg cisplatin or PBS. After 48 h, the bone marrow was isolated and analyzed as described below. Bone marrow from the femora was flushed out using a 21-gauge syringe with cold PBEA buffer (1 × PBS 0.5% BSA, 2 mM EDTA and 0.02% sodium azide). The samples were kept on ice. 10 × 10⁶ cells were used per staining. The following antibodies were used: Mouse Lineage Cell Detection Cocktail biotin antibody (1:40) followed by c-kit-APC (Clone 2B8, eBioscience), Streptavidin-APC-Cy7 (Southern Biotech), CD135-PE (Clone A2F10), CD48-PE-Dazzle (Clone HM48-1), 7AAD-PE-Cy5, Sca1-PE-Cy7 (Clone D7), CD34-FITC (Clone RAM34, Invitrogen, 1:100), CD127-BV421 (Clone A7R34), CD150-BV650 (Clone TC15-12F12.2), CD16/32-BV786 (clone 2.4G2, BD Bioscience). All the antibodies for FACS analysis were from Biolegend and used 1:200, unless otherwise specified. All measurements were performed with a BD LSRFortessa cell analyzer (BD Biosciences). Analyses were performed using FlowJo version 10.0.8r1.

Mice were euthanized at the indicated age (8–16 weeks) and BM cells from the femur and tibia were flushed out using 21-gauge syringes in cold PBEA buffer (1× PBS, 0.5% BSA, 2 mM EDTA, 0.02% Sodium Azide). Erythrocytes were eliminated from the BM cells using an erylysis buffer (NH₄Cl, KHCO₃, 0.5M EDTA) and passed through a 70 µm filter. Next, 10 million cells were used for staining. The following antibodies were used: Mouse Lineage Cell Detection Cocktail biotin antibody (1:40) followed by c-kit-APC (Clone 2B8, eBioscience, Sanata Clara, CA, USA), Streptavidin-APC-Cy7 (Southern Biotech, Homewood, AL, USA), CD135-PE (Clone A2F10), CD48-PE-Dazzle (Clone HM48-1), Sca1-PE-Cy7 (Clone D7), CD34-FITC (Clone RAM34, 1:100, Invitrogen, Waltham, MA, USA), CD127-BV421 (Clone A7R34), CD150-BV650 (Clone TC15-12F12.2) and CD16/32-BV786 (clone 2.4G2, BD Biosciences, Franklin Lakes, NJ, USA). Dead cells were excluded by 7AAD staining. All the antibodies for FACS analysis were from Biolegend, San Diego, CA, USA and used at 1:200, unless otherwise specified.