Moflo mls high speed cell sorter

For each experiment, retinas from two to four HRGP-Cre; R26-CAG-LSL-Sun1-sfGFP-myc mice were homogenized using a loose pestle in a Dounce homogenizer in ice-cold homogenization buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl₂, 20 mM Tricine-KOH, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine) with EDTA-free protease inhibitor (11 836 170 001, Roche, Basel, Switzerland). After addition of 5% IGEPAL-630 to bring the sample to 0.3% IGEPAL-630, the sample was further homogenized using a tight pestle. The sample was filtered using a 40 µm strainer (08-771-1, Fisher Scientific, Waltham, MA), mixed with 1.5 ml of 50% iodixanol density medium (D1556, Sigma, St. Louis, MO), and pelleted by centrifugation at 10,000g for 18 min in a swinging bucket centrifuge at 4°C. Nuclei were sorted using a MoFlo MLS high-speed cell sorter (Beckman Coulter, Brea, CA). Nuclei were sorted into either Buffer RLT for RNA preparation or PBS for DNA preparation (see below). An aliquot of nuclei sorted using the same parameters was placed into an additional tube. After sorting, this aliquot was inspected using a Zeiss Imager Z1 and Apotome system in order to verify the purity of the sorted sample.

Peripheral blood was taken from wing veins with heparin and washed twice with cold PBS by centrifugation at 300 g at 4°C for 5 min and resuspension in cold PBS. Cells were counted using a hemocytometer, and around 5 × 10⁷ lymphocytic cells in 2 ml were stained at 4°C in the dark for 1 h using T cell specific antibodies [10 µl mouse anti-chicken CD4-FITC and 10 µl of mouse anti-chicken CD8b-FITC for lines N and P2a; 10 µl mouse anti-chicken CD4-RPE and 10 µl of mouse anti-chicken CD8-RPE for lines 15I and 6₁ (all antibodies from Southern Biotech)]. Then the cells were washed 3–4 times with cold PBS and resuspended into 1 ml cold PBS for sorting, using magnetic-activated cell sorting (MACS, Miltenyi Biotec) for line N and P2a, and a DakoCytomation MoFlo MLS high-speed cell sorter (Beckman Coulter) for fluorescence-activated cell sorting (FACS, performed by Mr. Nigel Miller in the Department of Pathology) for line 15I and 6₁.

BAC aldh1l1-eGFP⁺ transgenic mice (P8, or P30-60) were used for FAC sorting at the Tufts FACS facility. Animals were deeply anesthetized with ketamine (100 mg/kg) + xylazine (10 mg/kg) in saline by i.p. injection and perfused intracardially with Hanks Buffered Salt Solution (HBSS) (Thermo Scientific). The brain was immediately dissected in cold Hanks buffer supplemented with glutamate receptor antagonists, 3 mM DNQX and 100 mM APV (Sigma, St. Louis, MO), and cut into small pieces. Cell suspension was prepared by following the manufacturer’s instructions in the neural tissue dissociation kit (Miltenyi Biotech, Auburn, CA). Briefly, small pieces of tissue were treated with papain enzymatic mix (37 °C, 15 min) and then digested with DNase I (37 °C, 10 min), followed by careful trituration. Cell mixtures were then filtered through a cell strainer (40–70 mm) and resuspended in cold HBSS solution (5–10 × 10⁶ cells/mL) for FACS. Cells were sorted by using MoFlo MLS high-speed cell sorter (Beckman Coulter) with Summit version 4.3 software. The whole procedure for cell suspension preparation and FAC sorting process was completed within 2–3 h. FAC-sorted cells were spun down and RNA was isolated from the cell pellet using standard TRIzol reagent.