Accumax

Monolayer cells and MTS after six days of culture were dispersed using Accumax (#SCR006 Chemicon) in EMEM medium with 5% fetal bovine serum and filtered through a 70 μm mesh (# 352350, Falcon Corning, Tewksbury, MA, USA). The cell cycle was determined by flow cytometry using the DNA Reagent Kit (Cycletest Plus #340242, BD, Billerica, MA) following the manufacturer’s recommendations. The samples were processed in a BD FACSAria Cell Sorter 3 and analyzed using the ModFit LT 3.2 version software. Each individual assay was performed in triplicate. Statistical comparisons were performed using t-tests.

Tumor tissues were finely minced and incubated in ACCUMAX™ (Chemicon) for 30 min at 37 °C. One volume of PBS was added to the cell solution, which was then centrifuged at 300 × g for 5 min. Supernatant was removed and cell pellets were resuspended in cold organoid medium (Neurobasal medium (Invitrogen) supplemented with 20 pg/mL EGF (R&D Systems), 10 pg/mL bFGF (Peprotech), 1X B27 (Invitrogen), 2.5% knockout serum replacement, 2.5% fetal bovine serum, 20 mM Glutamax, 1 mM sodium pyruvate, 0.25 µg/mL amphotericin B, and 100 U/mL penicillin-streptomycin). Tumor cell solution was embedded in Matrigel® (growth factor reduced, Corning) at a 1:1.8 ratio of cell solution to Matrigel® solution. A total of 20 µL mixed cell-gel solution was added to six-well plates via 5–7 drops/well and solidified in an incubator (37 °C) for 30–45 min. Organoid medium was added to cover the gel drops; cultures were maintained in a humidified incubator, with 5% CO₂, at 37 °C. RB organoids were manually dissociated and passaged at a 1:3 or 1:4 ratio every 3–4 weeks by embedding in fresh Matrigel®. Cold freezing medium (organoid medium containing 10% dimethylsulfoxide) was used to freeze organoids at −80 °C for 24 h prior to long-term storage in liquid nitrogen.

Cells grown as a monolayer and 6 days MTS were disaggregated with Accumax (Chemicon, MA, USA. #SCR006) were seeded and subjected to real-time migration monitoring using the CIM-Plate 16 and xCELLigence System using a RTCA DP Instrument (ACEA Biosciences, Inc, San Diego CA, 92121 USA). In this system, 40 000 cells of each culture condition were seeded in the upper chamber of a CIM-Plate 16 without FBS. The upper chamber was then placed on the lower chamber of the CIM-Plate 16 containing growth medium supplemented with 10% FBS as an attractant, medium without FBS (negative control). Cell migration was monitored over a period of 24 to 30 hours. MDA-MB-231 cells were used as a positive control (Fig. S1). Three assays were performed for each culture condition. Statistical analysis was performed using t-tests.

H9 colonies were dissociated by Accumax (Chemicon, Temecula, CA) and blocked with anti-human Fc- receptor (Miltenyi Biotec, Bergisch Gladbach, Germany). Following Fc blocking, cells were incubated with the phycoerythrin (PE)-conjugated p75 antibody (557196, BD Pharmingen, CA) for 20 min at 4°C. For HNK-1 and p75 double staining, the cells were firstly stained with HNK-1-FITC (322306, Biolegend, CA), then followed by conjugated anti-p75-PE. Compensation for FITC and PE was performed using compensation beads (BD Pharmingen, San Jose, CA). Positive and negative gates were determined using IgG stained and unstained controls.p75⁺or HNK-1⁺/p75⁺hNCSC cells were routinely maintained in self-renewal medium [14 (link)]on 6-well ultra-low attachment plates (Corning, Lowell, MA) at a density of 5×10³ cells/ml.

WT and Cstf2^E6 mESCs were prepared for staining by washing with warm Dulbecco's Phosphate-buffered Saline (DPBS) and detached using Accumax (Millipore). Cells were pelleted at 400 × g and washed with DPBS supplemented with 0.01% fetal bovine serum (FBS) followed by overnight fixation with 70% ethanol. Following fixation, cells were treated with 40 μg/ml RNase A (Thermo) for 30 min at 37°C and stained with 80 μg/ml propidium iodide (Life Technologies) for 1 h at 4°C. Stained cells were analyzed using a BD LSRII flow cytometer and cell cycle distribution was calculated using FlowJo software.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Cor.4U®, were acquired from Axiogenesis Inc. (Germany). These spontaneously beating cells represent a mixture of atrial, nodal and ventricular cardiomyocytes, with 60% being of the ventricular type. Cor.4U® hiPSC-CMs were delivered as fresh cells in T25-flasks (Nunc^©, Thermo Fisher Scientific) and kept in an incubator (5% CO₂, 37°C) and fed daily with Cor.4U® complete culture medium (Axiogenesis Inc.) supplemented with 1X Antibiotic-Antimycotic (Thermo Fisher Scientific) to prevent bacterial and fungal infection. For further passaging T25 flasks were pre-coated with 10 μg/ml fibronectin (Sigma) in PBS (with Ca²⁺ and Mg²⁺, Gibco®, Thermo Fisher Scientific) for 3 h at 37 or at 4°C o/n and the solution was removed shortly before plating the cells. For passaging, cells were detached using Accumax (Millipore) according to the manufacturer's instructions. The cells were collected by centrifugation (200 g, 3 min), the supernatant was removed and the cell pellet was gently re-suspended in the culture medium. Viable cells were counted using the trypan blue exclusion method and the cell density calculated according to viable cells. After plating, the cells were kept in the cell culture hood for 15 min to ensure that the cells settled evenly.