V450 mouse lineage antibody cocktail

To measure BM engraftment, mouse BM cells from femur/tibia were flushed by PBS and labeled for flow cytometry analysis. After Fc receptor block (BD Biosciences, San Jose, CA), bone marrow cells were stained at 4 °C for 15 min with the following antibodies (BD Biosciences): V450 mouse‐lineage antibody cocktail, PE‐Cy7 rat anti‐mouse Sca1, APC rat anti‐mouse cKit, PE mouse anti‐mouse CD45.1, and FITC mouse anti‐mouse CD45.2. After fixation with 2% PFA at 4 °C for 15 min, samples were ready for flow analysis.
To quantify the hematopoietic‐derived cells in the CNS, mice were perfused with 25 ml cold PBS before brain dissection. CNS‐infiltrated lymphocyte isolation was performed as described by Reddy et al. (2011). Briefly, isolated brains were treated with collagenase buffer at room temperature for 30 min after homogenization and passed through a 70‐μm cell strainer. The hematopoietic‐derived cells were separated on a Percoll (Sigma; GE17‐0891) gradient. Cells were then blocked with Fc receptor and stained with the following fluorophore‐conjugated antibodies: PE mouse anti‐mouse CD45.1 (BioLegend), FITC mouse anti‐mouse CD45.2 (BD Biosciences; 561874), and rat APC anti‐mouse CD11b (BD Biosciences; 553312). Fixed brain samples were used for flow analysis. All the BM and brain data were acquired using CyAn ADP (Beckman Coulter) and analyzed with Summit5.2.

BM was harvested from femurs by flushing with ice cold RPMI. The cell suspension was centrifuged at 800 g for 5 min, and supernatant aspirated. The cells were resuspended in RBC lysis buffer for 3 min and adding PBS with 1% FBS buffer and once again centrifuged. Cells were incubated with V450 mouse lineage antibody cocktail (561301, BD), in incubation with rat anti mouse Ly-6A/E (Sca-1) (558162, BD) and Rat anti mouse CD117 (c-kit) (553356, BD) antibodies, to quantify the percentage of Lin⁻ Sca1⁺cKit⁺ (LSK) Cells. Another population of cells from BM was stained with rat anti mouse CD34 (560238, BD) to identify the population of LT-HSCs and short-term (ST)-HSCs. The antibodies used for the MDSCs experiments were CD11b-PEcy7 (25-0112-82, eBioscience), Ly6G-eflur 450 (45-5931-82, eBioscience), and Ly6C-APC (17-5932-82, eBioscience). All antibodies were incubated for 30 min at 4°C and samples were analyzed with FACS Canto II (BD).