Acrodisc wbc filter

RBCs from donor B6 and HOD mice were obtained through cardiac puncture and collected in CPDA-1. RBCs were leukoreduced (Acrodisc WBC filter, Pall Life Sciences), washed with sterile PBS, then labeled with DiO (3,3′-dihexadecyloxacarbocyanine perchlorate, Molecular Probes Invitrogen, Carlsbad, CA, USA) or DiI (chloromethylbenzamido 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, Molecular Probes, Invitrogen) as previously described (25 (link)). In brief, HOD RBCs were resuspended in PBS to a 10% hematocrit and 100 uL of DiO (12.5 ug) was added for each 10 mL of RBCs. After labeling at 37°C for 30 min, RBCs were washed 3 times with PBS, and a sample was analyzed by flow cytometry to confirm labeling (Supplementary Figure 1). DiO fluorescence was read on the FL-1 (FITC) channel. B6 RBCs were diluted 1:14 in PBS (total volume 15 mL) and stained with 100 uL (50 ug) of DiI. B6 RBCs were incubated for 18 min at 37°C followed by another 12 min at room temperature, washed 3 times with PBS, and an aliquot was run on a flow cytometer to confirm labeling. DiI fluorescence was read on the FL-2 (PE) channel.