Ca2 and mg2

Spleens were minced into small pieces and digested in 1 mL of RPMI (Thermo Fisher Scientific) with 200 U/mL collagenase IV (Worthington) and 0.2 mg/mL DNAse I (Roche) for 30 min at 37 °C while shaking. After digestion, cells were passed through a 70 µm strainer and washed once with FACS buffer (phosphate-buffered saline (PBS), 1% fetal calf serum (FCS), 2.5 mM EDTA, 0.02% sodium azide). Erythrocytes were lysed with Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich) for 2 min at room temperature (RT), washed once, and resuspended in FACS buffer for further analysis. Bone marrow from adult mice was isolated from femurs and tibiae by flushing, and bone marrow from mice under 2 weeks of age was isolated by crushing the bones through a 70 μm cell strainer. Erythrocytes were lysed as above and cells were resuspended in FACS buffer for further analysis. Liver was minced into small pieces and digested in 2 mL PBS containing Mg²⁺ and Ca²⁺ (Sigma-Aldrich) with 1 mg/mL collagenase IV (Worthington), 60 U/mL DNAse I (Roche), 2.4 mg/mL Dispase II (Roche), and 3% FCS (Sigma-Aldrich) for 30 min at 37 °C while shaking. After digestion, cells were passed through a 100 µm strainer and centrifuged for 3 min at 50 g at 4 °C, to pellet hepatocytes. The supernatant was collected and recentrifuged for 7 min at 320 × g at 4 °C. Pelleted cells were resuspended in FACS buffer for further analysis.