2 mercaptoethanol

Sorted 4D4⁺ microglia 24 h post-TBI were cultured as previously described^{127 (link)} at 200,000 cells in a 24-well plate (Kemtec, cat. no. 4422A). The microglial culture medium was composed of 10% FBS (Gibco, cat. no. 10438026), 100 U ml⁻¹ of penicillin–streptomycin mixture (Lonza, cat. no. DE17-602E), supplemented in DMEM/F-12 Glutamax medium (Gibco, cat. no. 10565018). FoxP3-GFP was treated with nasal aCD3 or the isotype control for 7 d and the splenic/cLN CD4⁺FoxP3⁺ and CD4⁺FoxP3(GFP)⁻ population, specifically from aCD3-treated animals, was placed in a lymphocyte culture medium composed of 10% FBS, 100 U ml⁻¹ of penicillin–streptomycin mixture, 55 µM 2-mercaptoethanol (Gibco, cat. no. 21985023), 1% sodium pyruvate (Lonza, cat. no. BE13-115E) and 1% Hepes (Lonza, cat. no. BE17-737E), supplemented in RPMI-1640 medium (Gibco, cat. no. 11875119) and then placed on the top of the hanging cell culture 0.4-µm insert (Millicell, cat. no. PTHT24H48) at 600,000 cells per insert on top of the cultured microglia, and the assay was left for 72 h in a CO₂ Cell culture Incubator (InCusafe). There was a fourth group where IL-10-neutralizing antibody (BioXCell, cat. no. JES5-2A5) at a concentration of 50 μg ml⁻¹ was added with the CD4⁺FoxP3⁺ which was cell sorted from nasal aCD3-treated mice After 72 h the microglia were lysed with buffer RLT and RNA was extracted with RNeasy columns (QIAGEN).

mESC strains BRC6 (AES0010, RIKEN BRC) and ES-R1 (07072001, ECACC) were used in these experiments and maintained as described previously.⁹ (link) mESCs were cultured with mitomycin C (catalog no. M0503, Sigma-Aldrich)-treated SNL feeder cells (CBA-316, Cell Biolabs) in 5% CO₂ at 37°C in GKF medium (GKF ESM): Glasgow Minimum Essential Medium (catalog no. G6148, Sigma-Aldrich), 15% KnockOut Serum Replacement (catalog no. 10828-028, Thermo Fisher Scientific), 0.3% fetal bovine serum (FBS) (catalog no. 10437-028, Thermo Fisher Scientific), 2 mM L-glutamine (catalog no. 25030-081, Thermo Fisher Scientific), 1 mM sodium pyruvate (catalog no. 11360-070, Thermo Fisher Scientific), 1% MEM Non-essential Amino Acid Solution (catalog no. 11140-050, Thermo Fisher Scientific), 0.1 mM 2-mercaptoethanol (catalog no. 21985-023, Thermo Fisher Scientific), and 1% penicillin-streptomycin (catalog no. 15140-122, Thermo Fisher Scientific). For pluripotency maintenance, 1 μM MEK inhibitor (PD0325901) (catalog no. 162–25291, Wako), 3 μM GSK3β inhibitor (CHIR99021) (catalog no. SML1046, Sigma-Aldrich), and laboratory-prepared human LIF (1,000× dilution) were added to GKF ESM (2iL). Human LIF was produced in 293T cells by transfecting the pCAGGS-hLIF plasmid (RDB09182, RIKEN BRC DNA Bank).²⁵ (link) The titer of the supernatant containing human LIF was confirmed using BRC6 mESCs. mitomycin C-treated SNL feeder cells were prepared a few days before passaging mESCs in DMEM (catalog no. 10566-016, Thermo Fisher Scientific) containing 10% FBS and 1% penicillin-streptomycin on a gelatin (catalog no. G1890, Sigma-Aldrich)-coated plate/dish (1 × 10⁶ cells/10-cm dish). Guanine stock solution (20 mM) was prepared in 0.2 N NaOH.
To maintain mESCs, the culture medium was replaced with fresh medium daily. mESCs were dissociated by TrypLE Express (catalog no. 12604-013, Thermo Fisher Scientific) and counted using a counting chamber and Trypan Blue Solution (catalog no. T8154, Sigma-Aldrich) to passage the cells. The cells were passaged every 3 days at the density of 5 × 10⁴ (uninfected mESCs) or 1 × 10⁵ (infected mESCs cultured with puromycin) cells per well in 6-well plates (catalog no. 3516, Corning). To observe the cell growth as shown in Figure 1C, DsRedEx2-VSV-infected mESCs were passaged at the density of 2 × 10⁴ cells/well in 12-well plates (catalog no.3513, Corning). For Figures 2B and 2C, the DsRedEx2-VSV-infected mESCs after 39 days of culture with guanine and puromycin were passaged on a gelatin-coated 6-well plate at 2 × 10⁵ cells/well with or without guanine and puromycin. For the immunofluorescence staining images in Figure 1F, the infected and uninfected mESCs in pluripotent stem cell medium were passaged at 1×10⁴ cells/well on a gelatin-coated μ-Slide 8 well (catalog no. 80826, ibidi) and cultured for 3 days.
Nanog-VSV-infected mESCs (Figure 3B) were maintained in the presence of puromycin (10 μg/mL) and guanine (100 μM). For the transgene expression experiment, the cells were dissociated using TrypLE Express and washed with GKF ESM (-2iL) by centrifugation (200×g, 3 min) three times. The cells were then plated on a gelatin-coated 10 cm dish (catalog no. 3020-100, IWAKI) (6 × 10⁶ cells/dish) for flow cytometry analysis and a 6-well plate (1 × 10⁶ cells/well) for the detection of alkaline phosphatase activity in GFK ESM containing puromycin (1 μg/mL) with or without guanine (100 μM). Uninfected mESCs were cultured in parallel with the Nanog-VSV infected mESCs, which were plated on a gelatin-coated 10 cm dish (1.5 × 10⁶ cells/dish) for flow cytometry, and a 6-well plate (2.5 × 10⁵ cells/well) for the detection of alkaline phosphatase activity and cultured in GKF ESM (-2iL) without puromycin. The medium was changed daily during the 5 days of transgene induction period. The VSV vector removal experiments (Figures 4, S3, and S4) were performed using the mESCs infected with Nanog-VSV cultured in the differentiation induction medium after 5 days of transgene induction (Figure 3D, Guanine -, Infected (Exp1) and (Exp2)). Initially, the cells in each condition were passaged on mitomycin C-treated SNL feeder cells in a 6-well plate at 1 × 10⁵ cells/well in GKF ESM with 2iL and/or guanine (100 μM) and ribavirin (catalog no. S2504, Selleckchem) without puromycin. Ribavirin stock solution in water (10 mM) was added directly to GKF ESM with 2iL to the final concentrations of 10, 99, and 909 μM when the medium was replaced. The culture medium in each condition was replaced daily. Depending on the cell growth for each condition after the initial passage, the cells were passaged every 3 days at a cell density of 1 × 10⁵, 5 × 10⁴, or all cells per well in a 6-well plate and subsequently 3–4 × 10⁵, 2.5 × 10⁶, or all cells in a 10-cm dish.
Myogenic differentiation was performed following the schedule shown in Figure 5B. BRC6 mESCs infected with MyoD-VSV were maintained in GKF ESM containing 2iL, guanine (100 μM), and puromycin (10 μg/mL). The cells were dissociated by TrypLE Express and 1×10⁵ cells/well were passaged on a Biolaminin 521 LN (catalog no.BLA-LN521-05, BioLamina)-coated 12-well plate (1 × 10⁵ cells/well) (catalog no. 3513, Corning) for RNA extraction (day −1). For immunofluorescence staining, cells infected with MyoD-VSV were passaged on Biolamina 521-coated μ-Plate 24-well black (catalog no. 82406, ibidi) at 5 × 10⁴ cells/well (Figures 5E and S5C), or 24-well plates (catalog no. 3526, Corning) at 2 × 10⁴ cells/well (Figure S5D). The next day, the medium was replaced with fresh medium with or without guanine (100 μM) (day 0). The following day (day 1), the medium was replaced from GKF ESM to the serum-free differentiation induction medium containing DMEM (catalog no.12100-046, Thermo Fisher Scientific), 2% B-27 supplement (catalog no. 17504-044, Thermo Fisher Scientific), 2 mM L-glutamine, 1 mM sodium pyruvate, 1% MEM Non-essential Amino Acid Solution, 0.1 mM 2-mercaptoethanol, and 1% penicillin-streptomycin. The medium was adjusted to pH 7.6 under 5% CO₂ at 37°C using 1 M NaHCO₃. During myogenic differentiation induction, the culture medium was replaced daily until day 7. Puromycin (10 μg/mL) was added until day 5, and then the cells were cultured without puromycin to avoid the loss of differentiated cells.
For virus production, 293T (12022001, ECACC) and BHK-21 cells were maintained in DMEM (catalog no. 10566-016, Thermo Fisher Scientific) containing 1% penicillin-streptomycin and 10% FBS.