B220 apc ra3 6b2

WT or Smurf2-deficient mice (CD45.2⁺) were used as donors, and congenic CD45.1⁺ mice (The Jackson Laboratory, Bar Harbor, ME) were used as recipients. 8 to 10-weeks old Recipient mice were lethally irradiated (10 Gy) using a Cs¹³⁷ irradiator 24 hours before transplantation, and were treated with antibiotics (0.5 mg/ml neomycin and 100U/ml polymyxin-B) in drinking water 24 hours prior to exposure to radiation until 1 month after transplantation. For serial transplantation, 5×10⁶ donor BM cells were injected retro-orbitally into 8 to 10-week old lethally irradiated recipient mice. The transplantation cycle was repeated every 2 months.
For competitive repopulation, 2-month old WT CD45.1⁺/CD45.2⁺ mice were used as competitors. 1×10⁶ donor BM cells were mixed with 1×10⁶ competitor BM cells, and injected into 8 to 10-weeks old lethally irradiated recipient mice. The relative contributions from the donors, competitors or recipients in peripheral blood and BM of reconstituted recipients were analyzed in flow cytometry using antibodies to CD45.2-FITC (104) and CD45.1-PE-Cy7 (A20). Multi-lineage reconstitution in peripheral blood was analyzed using antibodies to B220-APC (RA3-6B2), CD3e-biotin (145-2C11) and CD11b-APC-Cy7 (M1/70, BD Biosciences). Antibodies were purchased from eBioscience unless specified.

Frozen sections from inguinal LNs were air dried, fixed for 10min in 2% PFA and washed in PBS. Blocking was performed for 30min at room temperature in 10% goat serum (Sigma) in PBS. Sections were stained for 1 hour at room temperature with B220-APC (RA3-6B2, BD Bioscience) and CD3-FITC (145-2C11, BD Bioscience) in blocking buffer. Images were acquired on a LSM710 upright confocal microscope (Zeiss) using a 10x objective and analyzed with Bitplane Imaris software.