Automated cell counter

docetaxel resistance assay was performed as previously described by Uzoh et al. [24 (link)], with slight modifications. PC3-luc cells with stable S6K knockdown or overexpression were seeded at a density of 8 × 10⁵ cells/well in 24-well plates and incubated for 24 h. The culture medium was then replaced by serum free media and the plates were incubated for 24 h. Afterwards, cells were treated with 30 nM of docetaxel (Sigma Aldrich) for 48 h, which was firstly dissolved in DMSO and then diluted in serum free media. Control cells received serum free media and the vehicle. The number of living cells was counted using an automated cell counter (Thermo Scientific) after Trypan Blue staining.

Adherent cells were washed three times with PBS and then treated in 1 mL of 0.25% trypsin–EDTA (Gibico, Waltham, MA, USA) followed by 3 min incubations at 37 °C. For suspension cells, the cell suspension was directly used for counting.
Trypsinized or suspended cells were mixed with trypan blue solution (0.4%) (Solarbio, Beijing, China) in equal volume and stained for 3 to 5 min prior to counting. Cell counting and viability assessment were performed with an automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA).

Cell proliferation assay was performed as previously described with minor modifications^{30 (link)}. Briefly, 5 × 10⁴ FTC-133 cells were seeded in six-well plates in medium supplemented with 1% FBS. Next day (day 0) cells were cultured in MRC-5 CM (8505c) or MRC-5 CM (KTC-2), replacing the MRC-5 CM (ATC) every 48 h (Fig. 7A). Nonattached cells were removed by gently washing twice with PBS. After trypsin treatment and resuspension, the number of attached cells was counted every 24 h from day 2–5 by using an automated cell counter (ThermoFisher Scientific).

Frozen cells were rapidly thawed at 37 °C and transferred into 50 mL centrifuge tubes. Subsequently, cells were counted using an automated cell counter (Thermo Fisher Scientific). An equal number of cells (3,300 per individual) from 4 different individuals were pooled together and loaded into a 10X Chromium Controller to generate Gel Beads-in-emulsion (GEMs). scRNA-seq libraries were generated according to the manufacturer’s instructions (Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) User Guide, Rev A, CG000315 Rev A). In brief, all the loading cells were separated into nanoliter-scale droplets. Within each droplet, cDNA was generated via reverse transcription, adding a cell barcoding sequence and unique molecular identifier (UMI) to each cDNA molecule. Library quality per pool was examined using the Agilent Bioanalyzer High Sensitivity DNA kit. Sequencing was carried out on NovaSeq 6000, having a 50,000 reads depth per cell. DNA was isolated from PBMCs and then subjected to genotyping by Illumina Global Screening Array Beadchip to demultiplex the pooled samples.

Organoids were kindly provided by Vivian Li’s lab. Crypts were isolated and plated in drops of basement membrane extract (BME) as previously described [23 (link)]. After BME polymerization, culture medium was added. The culture medium was prepared as described elsewhere [23 (link)], except that CHIR (R&D Systems) and RhoKinase inhibitor (Sigma) were also added.
For counting purposes, human organoids were single cell dissociated using TrypLE Express and mechanical dissociation with fire-polished Pasteur pipettes, after which they were counted using an Automated Cell Counter (Thermo Fisher).

NSCLC cell lines were seeded into 6-well plates at a density of 2 × 10⁶ cells per well and incubated with IFN-ɣ (100ng/ml). Untreated cells were used as a control. Following a 48 h incubation at 37 °C in a 5% CO₂ atmosphere, three random regions from both untreated and treated cells were captured (bars: 50 mm) with an inverted microscope Leica DM IL LED (Leica, Wetzlar, Germany). Cell viability was evaluated by 0.2% trypan blue exclusion test. Live cells were counted in duplicate with Automated Cell Counter (Thermo Fisher). Data are representative of the results obtained in three independent experiments.

After K562 cells were treated with 6AzGal and labeled with BDP-DBCO as described above, following cell assays were conducted. For trypan blue assay, 50 μL of cell suspension was mixed with equal volume of 0.4% (v/v) trypan blue solution, and then cell numbers were counted with Automated Cell Counter (ThermoFisher). For propidium iodide (PI)/annexin V-Alexa Fluor 488 apoptosis detection assay (ThermoFisher, A13201), 200 μL of cell suspension was transferred to 1.5 mL tube, mixed with 1 μL each stock solution of PI and annexin V, incubated at 25 °C for 15 mins, then analyzed with flow cytometry. For Cell Counting Kit-8 (CCK8) assay (Wako, 341-07761), 100 μL of cell suspension was inoculated on a 96-well plate per well. 10 μL of CCK8 stock solution was added into each well and incubated at 37 °C for 1 hour. The absorbance at 450 nm was measured with a plate reader.