Automacs cell separator

Human CD3⁺ lymphocytes as responder cells were isolated from spleens of humanized NSG mice using either a MACS human CD3 MicroBead Kit (Miltenyi Biotec, GlodBach, Germany) or an autoMACS™ Cell Separator (Miltenyi Biotec) according to the manufacturer's instructions. MLR assay was performed by previously reported methods^{8 (link)}. Briefly, stimulator cells were prepared from PBMCs of healthy human volunteers (n = 3) and irradiated at 30 Gy. Then 2 × 10⁵ hCD3⁺ responder cells purified from the humanized mice were plated in triplicate into a 96-well U-bottom plate and incubated without or with irradiated 4 × 10⁵ stimulator cells. After 3 days, MTT assay was performed to check cell proliferation according to the manufacture's instruction (Roche Diagnostics, Indianapolis, IN).

For the preparation of donor murine lung cells, E13.5, E15.5, E18.5 fetal lungs and P7, P14, P56 mouse lungs were collected and minced with a sterile razor. Minced lungs were dissociated with Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) with 0.96 mg/mL dispase II (Roche), 0.2% collagenase (Wako Pure Chemical Industries), and 20 kU/mL DNase I (Sigma-Aldrich) solution as previously described^{39 (link)}. After a 60-min incubation at 37 °C, cells were filtered through a 70-µm cell strainer (BD Biosciences), and washed with DMEM supplemented with 2% fetal bovine serum. Erythrocytes were removed by Percoll gradient (70%) centrifugation (GE Healthcare) for 20 min at room temperature (1,000 × g). Single-cell suspensions were further incubated with Ter119-biotinylated (1:200; clone TER-119; BD Biosciences) antibody for 20 min (4 °C), followed by anti-biotin MACS beads (Miltenyi) for 20 min (4 °C). Incubated cells were depleted of erythrocytes through negative selection using an AutoMACS cell separator (Miltenyi Biotech). Resulting propidium iodide staining negative cells were counted using Flow-Count fluorospheres (Beckman Coulter) and a Gallios flow cytometer (Beckman Coulter).

CD4⁺ T cells were enriched from the spleen and lymph nodes of control (Ezh2^fl/fl) and Ezh^−/− (Cd4 Cre; Ezh2^fl/fl) mice with an AutoMACS cell separator (Miltenyi Biotec), stained with PerCP Cy5.5 anti-CD4, FITC anti-CD25, and PE anti-CD45RB (all obtained from BD Biosciences), and naive CD4⁺ CD25⁻ CD45RB^hi T cells were purified (>99%) by cell sorting (Moflo,Dako Cytomation or FACSAria, BD Biosciences). CD4⁺GFP⁺CD25⁺NRP1⁺ tTreg cells were purified from Ezh2^fl/flFoxp3-GFP mice or Cd4 Cre; Ezh2^fl/flFoxp3-GFP. CD4⁺CD45RB^hiCD25⁻ naive T cells (4 × 10⁵) from control mice and Ezh2-deficient mice were injected intravenously (i.v.) into age- and sex-matched Rag2^−/− mice and intestinal inflammation was monitored. Alternatively, Rag2^−/− mice were coinjected with 4 × 10⁵ naive T cells and 1 × 10⁵ CD4⁺CD45RB^loCD25⁺ Treg cells from control mice and Ezh2-deficient mice. Mice were sacrificed at 7 weeks when significant weight loss occurred in the control groups. Gut cells were isolated as described previously by intracellular staining. Sections of the proximal, mid-, and distal colon were fixed in buffered 10% formalin and stained with hematoxylin and eosin (H&E) (Histoserv).

A three-stage differentiation protocol was used to generate heterogeneous cultures containing lens cells (Yang et al., 2010 (link)). Growth factors were sourced from Miltenyi Biotec and Peprotech, and the base medium for each stage was DMEM:F12 (Thermo Fisher Scientific). Initial modification of this protocol involved increasing the concentration of noggin to 500 ng/ml and including 10 nM SB431542 in the stage 1 medium, followed by reducing the concentration of FGF2 to 10 ng/ml in stage 3. For purification of ROR1⁺ cells via magnetic cell separation, single-cell suspensions were obtained using TrypLE (Thermo Fisher Scientific) during stage 2 of the lens differentiation protocol. The cells were then incubated with a biotinylated anti-human ROR1 antibody (BioScientific; AF2000) and labelled cells purified using anti-biotin microbeads and an autoMACS cell separator (Miltenyi Biotec). Purified ROR1⁺ cells were plated on Matrigel-coated dishes in M199 medium (Thermo Fisher Scientific) containing 10 ng/ml of FGF2 or in test media (TM) consisting of M199 and combinations of the following growth factors: BMP4/BMP7 (20 ng/ml each); EGF/TGFα (5 ng/ml each); HGF (10 ng/ml); IGF1/insulin (10 ng/ml and 10 µg/ml); and PDGF-AA (10 ng/ml). All four human pluripotent stem cell lines (two embryonic and two induced pluripotent) tested behaved similarly.