Tube rotator

Mice were anesthetized by inhalation of isoflurane then perfused with 30–50 mL of ice cold PBS through the left ventricle. Brains were collected and minced with scissors before treatment with Liberase TL (Roche, Indianapolis, IN) and DNAse I (Invitrogen, Carlsbad, CA) at 37° C on a tube rotator (Miltenyi Biotec, Cambridge, MA) for 30 minutes. Samples were pushed through 100µM Nytex Cell Strainers (BD Biosciences, San Jose, CA) to yield a single cell suspension. Mononuclear cells were isolated using a 30%/70% Percoll gradient (Amersham, Piscataway, NJ) centrifuged at 2500×g for 20 min at room temperature without break. Cells were counted using Tryphan Blue exclusion (Life Technologies Invitrogen, Carlsbad, CA). Spleens were collected from mice at time of perfusion. Spleens were mashed and red blood cells were lysed using NH₄Cl to obtain a single cell suspension of spleen infiltrating lymphocytes.

After euthanasia, animals were perfused by injecting 10 ml of ice-cold PBS into the left ventricles, and hearts and livers were collected to isolate mononuclear cells (MNCs) as reported previously (Gangaplara et al., 2012 (link); Bharathi Krishnan et al., 2017 ). In brief, livers were minced, and the dissociated tissues were treated with Collagenase IV (400 U/ml; Worthington, Lakewood, NJ) and DNase I (150 U/ml; Worthington) at 37°C in shaker incubator for 30 minutes. After washing, MNCs were harvested using 40%/75% percoll gradient centrifugation procedure as described previously (Bharathi Krishnan et al., 2017 ). After lysing erythrocytes, cell pellets were dissolved for further experimentation. For isolation of MNCs from hearts, the minced tissues were treated with Collagenase II (600 U/ml; Worthington Biochemical Corporation, Lakewood, NJ, USA) and DNase I (60 U/ml; AppliChem GmbH, Darmstadt, Germany) for 30 minutes at 37°C using tube rotator (Miltenyi Biotec Inc., Auburn, CA). Cell suspensions were filtered through 70 μM cell strainer, and after removing the residual debris using debris removal kit (Miltenyi Biotec) and lysing erythrocytes, MNC suspensions were obtained.