H 2kb apc

After 10 passages, the cells were characterized by multiple commonly utilized markers for conventional MSCs. Phenotypic antibodies used are as follows: CD45-FITC, CD90-FITC, CD34-PE, CD11c-FITC, CD44-PE, I-A^b-FITC, CD80-FITC, CD11b-PE-Cy7, CD73-PE (all from BD Biosciences) and H-2k^b-APC, CD105-PE, Sca-1-FITC, CD9-PE, and CD86-PE (all from eBioscience). All isotype controls were purchased from BioLegend. Flow Cytometry was conducted on a BD FacsCalibur. Standard protocols were conducted to verify the ability of MSCs to differentiate into adipocytes and osteoblasts using Mesencult mouse adipogenic stimulatory supplements and mouse osteogenic stimulatory supplements (StemCell Technologies products), respectively, in IMDM-based media with 10% FBS supplementation. Supplement-to-basal media was at a 1:4 ratio. For both differentiation tests, 2.5×10⁴ MSC were plated per well in 2mL Mesencult media in 6-well plates in 37°C/ 10% CO2 incubation. On day 3, cells were given appropriate differentiation media, with media changes every 3– 4 days. After 2 weeks of culture, cells were stained for adipocytic differentiation with Oil Red O and osteogenic differentiation with Alizarin red. Human MSCs were phenotyped by Lonza as CD105+CD166+CD29+CD44+ > 90% and CD14+CD34+CD45+ <10%.

Mice were bled at 4 weeks of life by retro-orbital venipuncture using Heparinized Micro-hematocrit Capillary Tubes (Kimble Chase Cat#40C505) under isoflurane anesthesia. Red cell lysis was performed with BD Pharm Lyse (BD Biosciences Cat#555899). Cells were stained with CD45-PE (Biolegend Cat#103106), H2k^b-APC (eBioscience Ref#17–5958-82), and H2k^d-PerCP/Cy5.5 (Biolegend Cat#116618) at 1:100 for 25 minutes at 4°C. Donor cell chimerism was assessed among CD45⁺ cells and was calculated as (H2k^b+GFP⁺ / (H2k^b+GFP⁺ + H2k^d+)) × 100 for mice injected with B6^GFP BM and as (H2k^b+ / (H2k^b+ + H2k^d+)) × 100 for mice injected with B6 BM.