Triple express enzyme

Control (n = 3) and D164A (n = 3) mice have sacrificed 0, 2 or 4d post injection, crypts were isolated as described^{50 (link)}. Crypt fractions 3 and 4 were pooled and dissociated to single cells by incubating them with 5 mL pre-warmed TripLE Express Enzyme (ThermoFischer) in a 37 °C water bath for 5 min with frequent agitation. TripLE was inactivated with 50% FBS in Advanced DMEM/F12 (ThermoFischer). Single cells were pelleted at 180 × g for 3 min and resuspended in 2 mL cold Intesticult (Stemcell Technologies Inc). Clumps were dissolved by pipetting up and down with a 1 mL pipet before filtering twice through a 40 μm filter. Cell viability and number were quantified in automatically with a Countess (Thermo Scientific). DropSeq workflow was performed as described⁶² on a Nadia Instrument (Dolomite Bio). Sequencing was performed on the Illumina NovaSeq6000 with an SP Reagent Kit.

The hiPSCs were maintained in 10 cm plastic plates (Corning Inc., NY, USA) coated with 0.5 μg/cm² iMatrix511 Silk (Nippi Inc., Tokyo, Japan) with StemFit^® AK02N (Ajinomoto, Tokyo, Japan) as the culture medium under a controlled atmosphere at 37 °C, 5% CO2, and > 95% humidity. The cells that reached 70–90% confluency were washed three times with phosphate-buffered saline without calcium (PBS(-); Nacalai Tesque Inc., Kyoto, Japan) and treated with 3 ml of TripLE™ express enzyme (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 μM of the Rho kinase inhibitor Y-27632 (Sellec Inc., Tokyo, Japan) for 15 min at 37 °C. Detachment and dispersion into single cells were performed by pipetting using 10 ml pipettes with the addition of 5 ml of PBS(-) supplemented with 0.15% bovine serum albumin fraction V (BSA: Fuji Film Wako Chemical Inc., Miyazaki, Japan). The cells were collected in a 15 ml tube and centrifuged at 800 rpm (115× g) for 5 min for deposition. The supernatant was then aspirated, and the cell pellet was dispersed into single cells using 10 ml Stemfit supplemented with 10 μM Y-27632 by pipetting.

The cancer cell line U-87 MG was sourced from the Russian cell culture collection (Russian Branch of the ETCS, St. Petersburg, Russia). U-87 MG cells were grown in α-MEM with nucleosides (Gibco, Waltham, MA, USA), supplemented with 10% of fetal bovine serum (Gibco, Waltham, MA, USA), 1 mM L-glutamine, and 1% (v/v) antibiotic–antimycotic solution (Gibco, Waltham, MA, USA). Cells were cultivated in 25 cm³ tissue culture flasks in a humidified 37 °C incubator supplied with 5% CO₂ and were passaged with the TripLE Express Enzyme (Thermo Fisher Scientific, Waltham, MA, USA) every 3–4 days.