Igg2a clone rg7 1.30

DSA in humans were analyzed as described previously, using the single antigen bead Luminex (One Lambda) assay (23 (link),24 (link)). Rat DSA measurement was performed as described elsewhere (25 (link),26 (link)). Briefly, donor (BN, RT1ⁿ) splenocytes were freshly isolated from spleen macerated through a 50-μm sieve and washed after red blood cell lysis. Cells were resuspended, counted and aliquoted into cluster tubes for staining. Fifty microliters of 1:4 dilution of serum or plasma from 1 week posttransplant (or similar time points for control or transfusion only rats) were incubated for 30 min at 37°C, then washed and stained. Flow cytometry was performed on a BD LSR II at the Immunology Core of the WNPRC, UW-Madison and data analyzed using FlowJo (TreeStar, Inc., Ashland, OR). Cells were gated to remove nonsinglets, through a lymphocyte gate, then a CD3-positive gate to remove cells that had an FcR, which could skew analyses. Mean fluorescence intensity was determined for the populations of interest. Antibodies used were anti-IgG1 (clone RG11/39.4; BD Biosciences, San Diego, CA), IgG2a (clone RG7/1.30; BD Biosciences), IgG2b (clone RG7/11.1; BD Biosciences), IgG2c (biotinylated, clone A92-1, BD Biosciences), IgM (clone G53-238; BD Biosciences) and CD3 (clone 1F4 [BioLegend, San Diego, CA] or clone G4.18 [BD Biosciences]).

Recipient DSA measurement was performed as described previously.^{30 (link)} Donor splenocytes were incubated with recipient plasma, staining was measured by flow cytometry (BD LSR II), and analyzed using FlowJo (TreeStar, Inc., Ashland, OR). Antibodies used were directed against: IgG (112-096-003, Jackson ImmunoResearch), IgG1 (clone RG11/39.4, BD Biosciences, San Diego, CA), IgG2a (clone RG7/1.30, BD Biosciences), IgG2b (clone RG7/11.1, BD Biosciences), IgG2c (biotinylated clone A92-1, BD Biosciences), IgM (clone G53-238, BD Biosciences), and CD3 (clone 1F4, BioLegend; or clone G4.18, BD Biosciences).