G7513

Bra-GFP/Rosa26-E2C mESCs (Zhou et al., 2018 (link)) were maintained in 0.1% gelatinised six-well tissue culture plates with mitomycin-C (Sigma-Aldrich, M4287) inactivated STO (ATCC, SCRC-1049) feeder cells at 37°C in a humidified incubator with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, D6546) supplemented with 15% fetal bovine serum (FBS) (Sigma-Aldrich, F2442), 1% minimum essential medium (MEM) non-essential amino acid (Sigma-Aldrich, M7145), 2 mmol l⁻¹ L-glutamine (Sigma-Aldrich, G7513), 0.1 mmol l⁻¹ β-mercaptoethanol (Gibco, 31350) and 1000 U ml⁻¹ mouse leukaemia inhibitory factor (mLIF) (Merck Millipore, ESG1107). Cells were passaged every other day and those at passage 13–22 were used for experiments.
GFP-expressing mouse neonatal kidney-derived stem cells (GFP-KSCs) (E. Ranghini, PhD thesis, 2011) were maintained in 60-mm tissue culture dishes at 37°C in a humidified incubator with 5% CO₂ in DMEM supplemented with 10% FBS (Gibco, 10270), 1% MEM non-essential amino acid (Sigma-Aldrich, M7145), 2 mmol l⁻¹ L-glutamine (Sigma-Aldrich, G7513) and 0.1 mmol l⁻¹ β-mercaptoethanol (Gibco, 31350). Cells were passaged 2–3 times per week and those at passage 17–20 were used for experiments.

Mice were immunized as previously described (see Section 2.7). Eighteen days after the boost vaccination, spleens were harvested from the mice and were pushed through a 70-µM strainer (Foxx Life Sciences, Salem, NH, USA). The splenocytes were then centrifuged at 1600 rpm for 5 min (HERAEUS Megafuge16, Thermo Scientific, Waltham, MA, USA), and red blood cells were lysed in ammonium-chloride-potassium (ACK) lysis buffer comprised of 0.15 M NH₄Cl (Sigma A-4514, MW = 53.49), 10 mM KHCO₃ (Sigma P-9144), and Na₂EDTA (Sigma ED2SS), pH 7.2. Splenocytes were then centrifuged at 1600 rpm for 5 min and were resuspended in complete α-medium (MEM α-modification; Sigma M4526, 10% FCS; Sigma F-2442, 100 U penicillin/100 µg streptomycin mix; Sigma P-0781, 4 mM l-glutamine; Sigma G-7513, 50 µM 2-Mercaptoethanol; Gibco BRL 31350-010) at a cell concentration of 2.5 × 10⁶ cells/mL.

All cell lines were obtained from the American Type Culture Collection (ATCC) and cultivated in a humidified incubator at 37 °C and 5% CO₂. The human keratinocyte cell line HaCaT was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, D6419) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, F7524), 2 mM glutamine (Sigma-Aldrich, G7513), 0.1 mg/ml gentamicin (Gibco, 15710049), and 1.25 µg/ml fungizone (Sigma-Aldrich, A2942). For the lung carcinoma cell line A549 we used DMEM supplemented with 10% FBS and 2 mM glutamine, while the colorectal carcinoma cell line LS411N and the colorectal adenocarcinoma cell line DLD1 were cultivated in RPMI 1640 medium (Gibco, A1049101) supplemented with 10% FBS.