H2kb pecy7

In order to determine the impact of maternal IgG on fetal T cell sensitization, an in vivo mixed lymphocyte reaction (MLR) was performed. Lymph nodes and spleens were harvested from experimental animals, and LDMCs were isolated as described above. Adult Balb/c mice injected twice intraperitoneally with 20 × 10⁶ B6 splenic LDMCs 1 and 2 weeks prior to the MLR served as positive controls for T cell sensitization to donor cell antigen. Cells were stained with Cell Proliferation Dye eFluor 670 (eBiosciences Cat #65-0840-85, fluorescence equivalent to APC) at a concentration of 1:250 in 1mL cRPMI 1640 medium (Gibco Cat #11875093) for 40 minutes at 37 °C. Cells were gently agitated once after 20 mins. The suspension was washed with 49mL ice-cold PBS and resuspended in 200μL PBS. Cells were then injected via the tail vein into 8-week-old B6 × Balb/c F1 offspring (H2k^bH2k^d). After 72 hours, F1 mice were sacrificed, and the lymph nodes and spleens were isolated separately. LDMCs were stained with CD3-PE (BD Biosciences Cat #555275) and H2kb-PECy7 (eBiosciences Cat #25-5958-82) at a concentration of 1:100 in staining buffer for 30 mins. The percent proliferating was determined by flow cytometry for host T cells as the percentage of H2k^b-GFP^-CD3⁺ cells with APC fluorescence intensity ≤50% of undivided cells.