Isolation of Mononuclear Cells from Blood, Heart, and Spleen

Mononuclear cells were isolated from the peripheral blood, heart, and spleen as previously described [16 (link)]. Peripheral blood (~200 μL) was collected in EDTA tubes (BD Biosciences). Erythrocytes were lysed with RBC lysis buffer (eBioscience) and the remaining leukocytes were washed with PBS, collected by centrifugation (380g for 10 min at 4°C) and resuspended in 400 μL ice-cold flow cytometry staining buffer (eBiosciences). Splenocytes were isolated by flushing the spleen with 3mL of PBS and then filtering the cells through a 100 μm cell strainer (BD Falcon). Cells were centrifuged at 500g for 5 min at 4°C and resuspended in staining buffer (eBioscience). For cardiac immune cell isolation, whole hearts were flushed with PBS to remove blood and minced into 2mm pieces using a single-edged blade. The tissue was digested in RPMI media containing collagenase-2 (1mg/mL, Worthington), trypsin (1 mg/mL, Invitrogen), and DNase I (10 ug/mL) at 37°C for 45 min with gentle agitation. Cell suspensions were filtered through a 100 μm cell strainer (BD Biosciences) and incubated with 2 mM EDTA in PBS for 5 min at 37°C. Isolates were then centrifuged at 2000g for 20 min on a Ficoll gradient (GE Healthcare), and mononuclear cells were subsequently collected and washed with PBS. Cells were fixed with 1% paraformaldehyde and stored in staining buffer (eBioscience) at 4°C.