Tryple express

hESC colony cultures were incubated with the Rho Kinase inhibitor Y-27632 (Rock Inhibitor, 10 μM: Sigma-Aldrich, St Louis, MO, USA) for 1 h and dissociated to single cells using TrypLE™ Express. Cell aggregates (1000–1500 cells) were formed using the AggreWell™400 system (StemCell Technologies) and cultured in Basal Differentiation Media (BDM) with Rock Inhibitor (Table S1). For differentiation, aggregates were cultured in BDM supplemented with combinations of SB43218 (SB: 10 μM), CKI-7 (CKI: 5 μM) and nicotinamide (NIC: 10 mM) (all Sigma-Aldrich). After 6 days, embryoid bodies (EBs) were plated on GFR-MG-coated plates in BDM.
At day 8, expanding EBs were dissociated with collagenase IV (1 mg/mL) and re-plated as clumps of 200 to 500 cells on GFR-MG-coated 24-well dishes. Cells were then cultured in BDM without factors and refed every 2 to 3 days until day 38.

Dulbecco’s modified Eagle medium (DMEM) powder was purchased from Gibco by Life Technology (Thermo Fisher Scientific, USA). Calcium chloride dihydrate (CaCl₂∙2H₂O), sodium bicarbonate (NaHCO₃), sodium hydrogen phosphate (Na₂HPO₄), potassium dihydrogen phosphate (KH₂PO₄), sodium citrate tribasic dihydrate, and alpha-ketoglutaric acid disodium salt hydrate were obtained from Sigma-Aldrich (St Louis, MO, USA). Anticancer drugs DOX and CYP were acquired from Sigma-Aldrich (St Louis, MO, USA). DMEM liquid media, fetal bovine serum (FBS), trypsin, TrypLE Express, penicillin–streptomycin, and trypan blue were procured from Sigma-Aldrich (St Louis, MO, USA). Sodium chloride and potassium chloride salts were bought from Fischer Scientific (Loughborough, UK). Acetonitrile (ACN), hydrochloric acid (HCl), and methanol were from Fischer Scientific (Loughborough, UK). Ammonium bicarbonate, formic acid, dithiothreitol (DTT), trifluoroacetic (TFA), trifluoroethanol (TFE) acid, and iodoacetamide (IAM) were from Sigma-Aldrich (St. Louis, MO, USA).

Mitoxantrone (MTX) was purchased from Sigma-Aldrich (M6545) and used at a concentration of 8 µM to induce immunogenic cell death. Apoptosis induction was counteracted by pre-treating for 4 h with the pan-caspases inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-fmk, 50 μM) purchased from Bachem (N1560).
To estimate the amount of cell death, supernatant and cells were collected with TrypLE Express and resuspended in PBS containing propidium iodide (PI) (Sigma, P4170). Samples were subsequently acquired via Attune Flow Cytometer (Life Technologies) and data analysis was performed via FlowJo_V10^™ software (Tree Star, Ashland, OR, USA).

Freshly harvested tumours were dissected in DMEM/F12 (2% PS), followed by incubation in 5 mM EGTA-PBS at 4 °C, shaking gently, to remove potential contaminant cells from the normal tissue. Tumours were then minced in small pieces with razor blades and incubated in diluted TrypLE Express in PBS (66%), shaking (180 rpm) for 45 minutes at 37 °C. Trypsin was inactivated with 10% of cold FBS. The cell suspension obtained was then filtered through a 40 µm cell strainer and cells were counted upon centrifugation for 5 min at 450 g, following re-suspension in Flow buffer (DMEM/F12, 5 mM EDTA, 1% BSA, 1% FBS, 10U/ml DNAse (D4263-5VL Sigma-Aldrich)). Small intestines were harvested and flushed with cold 1X PBS, followed by longitudinal opening and cut into small pieces of approximately 5 mm × 5 mm. Intestinal fragments were subsequently incubated with 2 mM EDTA in HBSS for 30 minutes at 4 °C. Crypts were obtained by serial fractioning. Optimal crypt isolation was checked under the microscope. Crypts were then incubated in TrypLE Express (ThermoFisher Scientific) for 5 minutes at 37 °C to obtain single cells. TrypLE Express was inactivated with 10% cold FBS (Sigma-Aldrich). The cell suspension obtained was filtered through a 40 µm cell strainer and counted cells were resuspended in Flow buffer.

Cells were disaggregated with TrypLE Express and fixed in 2% paraformaldehyde (Sigma-Aldrich, Darmstadt, Germany). The cell membrane was permeabilized in 100% methanol at −20 °C for 10 min, washed once with DPBS (Biolot, Saint-Petersburg, Russia), and stained with the first antibodies diluted in 1% BSA in DPBS at 4 °C overnight. The next day, cells were washed once with DPBS, and secondary antibodies diluted in 1% BSA in DPBS were added for 1 h. Secondary antibodies were used as isotype controls. A cell count was performed by the BD FACSAria™ III (BD Biosciences, Franklin Lakes, NJ, USA) using the BD FACSDiva software. All the measurements were made using four replicates. The list of antibodies used in the FACS analysis is presented in Table 3.