Trypan blue 0.4

HTEpC (135,000 cells) were plated into three groups. Group 1 was transfected with CoV-229E (n = 3, 50uL per plate). In group 2, prior to transfection, CoV-229E was exposed to NB-UVA (n = 3, 2000 μW/cm²) for 20min. Group 3 was not exposed to NB-UVA or transfected (n = 3). After transfection, the cells were exposed to NB-UVA (4cm distance with 2000 μW/cm² at the plate surface) for 20min daily. Plates were imaged at 16, 36, 72, and 96hrs, cell viability (live/dead) counts were obtained at 72 and 96hrs post-transfection. Trypan Blue 0.4% (1:1) (Gibco) was used to determine live/dead cells and cell counts were obtained using an automated cell counter (Biorad T20, Hercules, CA). Cells were kept at 37ºC (5% CO₂).

A dye exclusion test was used to determine the viability of trophozoites after DMSO or ASA treatment, about 10 μL of culture was mixed with 10 μL trypan blue 0.4% (Gibco-BRL, Gaithersburg, MD, USA). The total number of parasites (including those which had excluded the dye), was counted in a Neubauer chamber and cell viability was calculated as the percentage of viable cells in the samples relative to untreated cells.

Clonal spontaneous growth is defined by the following parameters: the rate at which cells are born (birth rate, b), the rate at which cells die (death rate, d) and the net growth rate b − d. To estimate the b − d rate, CRC cell clones were seeded at 3.5–4.0 × 10⁵ cells per well in 6-multiwell plates. Plates were incubated at 37 °C in 5% CO₂. Starting from the next day, the number of viable cells was assessed by manual count in Trypan Blue 0.4% (Gibco) by two operators independently at the indicated timepoints, to obtain the clones’ net growth rate. To estimate d/b, cells were seeded at different densities (3.5–4.0 × 10⁵ cells per well) in multiple 6-multiwell plates. Plates were incubated at 37 °C in 5% CO₂. At each timepoint, cells were collected and stained with propidium iodide (Sigma-Aldrich) following the manufacturer’s instructions. Cells were then analyzed by flow cytometry. Data were acquired with the Beckman Coulter CyAn ADP instrument using the Summit v.4.3 software and analyzed with the FlowJo software (v.7.6). The cells in the sub-G1 phase were considered dead and used to estimate d/b. The values of birth and death rates were then obtained by combining b − d and d/b estimates (Supplementary Note).

HeLa cells were cultured (12 plates, mean 253,000 cells/plate) for 24hr at 37ºC (5% CO₂). Recombinant coxsackievirus B (pMKS1) expressing an enhanced green fluorescent protein (EGFP-CVB) was prepared as previously described [17 (link)]; half were exposed to NB-UVA (2000 μW/cm²) for 20min while the other half were not exposed. HeLa cells were then transfected with NB-UVA-exposed or NB-UVA-unexposed virus (multiplicity of infection (MOI) = 0.1). Coxsackievirus is considered highly lytic [18 (link)]. After 6hrs, supernatant was removed, and cells were washed twice with 1x sterile PBS (pH = 7.0). New DMEM media was added and cells were incubated at 37ºC (5% CO₂). Dead cells in the supernatant (floating cells) were collected and quantified 24hrs later using an automated cell counter (Biorad T20). Six plates (3 NB-UVA-exposed and 3 unexposed) were assessed for live cells. Of the remaining six plates, the 3 plates transfected with NB-UVA-exposed virus were exposed to an additional 20min of NB-UVA (2000 μW/cm²). After 24hrs at 37ºC (5% CO₂), imaging was performed using a BZ-9000 BioRevo (Keyence Corp., Itasca, IL). Dead and live cells were determined by the Trypan Blue 0.4% (1:1)(Gibco) method and counts were obtained using an automated cell counter (Biorad T20).

To verify both the biocompatibility of MOC platform and assess viability of Human Glioblastoma A-172 and HUVEC cells after μG experiments, live imaging using LIVE/DEAD Viability/Cytotoxicity Kit (Sigma-Aldrich, Cat. No. L3224) was performed as per manufactures instructions. Briefly, fluorescent calcein-AM (2 μM) and red-fluorescent ethidium homodimer-1 (EthD-1) (4 μM) were gently injected into the MOC and incubated for 15 min at 37 °C and humidify atmosphere. MOCs were then washed with PBS and imaged using EVOS M5000 microscope (Invitrogen). To evaluate proliferation, cells were detached from the MOC using Trypsin-EDTA, collected, centrifugated and counted using a Hemocytometer. Trypan Blue 0.4% (Thermofisher Scientific, Cat. No. 15250061) was used to determine the percentage of viable cells present in the suspension.