Celltiter glo luminescent viability assay

For PDX cell viability assays, PDX cells were plated in 96-well plates at 25,000 cells per well in M87 medium^{33 (link)} and treated with drugs for 72 h, followed by imaging and measurement of luciferase activity (total photon flux per second) two minutes after the addition of D-luciferin (15 mg/ml; GoldBio) to each well (1/10 of total volume per well), using the IVIS Spectrum In Vivo Imaging System (Xenogen IVIS-200) and living image software (PerkinElmer), as described in our previous work^{36 (link)}. For cell line viability assays, MDA468, HCC1143, or HCC1937 cells were plated in 96-well plates at 5,000 cells per well in complete RPMI-1640 GlutaMAX medium, cultured overnight to allow for adherence, and subsequently treated with drugs for 72 h. Viability of cell lines was measured using the CellTiter-Glo Luminescent Viability Assay (Promega), according to the provided protocol.

In the absence or presence of bortezomib (7.5 nM for MM1S, RPMI8226, and U266 cells, 30 nM for H929 cells), MM cells (3×10⁵ cells/mL) were treated with or without BMSC-derived sEVs for 48 h and the viability was measured with the Cell Titer glo Luminescent Viability assay (Promega, Madison, WI, USA). MM cells were treated with or without endocytosis inhibitors for 4.5 h and the cell viability was determined using the Cell Titer glo Luminescent Viability assay. All viability assays were performed in triplicate and repeated in three independent experiments. For apoptosis analysis, the treated cells were stained with Annexin V-FITC (Biolegend, San Diego, CA, USA) and 7-Aminoactinomycin D (7-AAD, Biolegend) and apoptotic cells were determined using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA).

A total of 4000 cells/well were seeded onto 96-well plates one day before and then the indicated concentrations of drugs were used to treat the cells for 48 h. CellTiter-Glo Luminescent Viability Assay (Promega) was used to monitor the cell viability according to the manufacturer’s instructions. A total of 250 cells were seeded onto a 35 mm dish one day before for the colony-forming assay. The cells were then treated with PBS or cisplatin for 48 h and then maintained in a fresh complete medium for 10 more days for colony formation. Crystal violet (0.5%) was used to stain the colonies. The number of cell colonies was calculated by ImageJ software 1.53v.

The GM00038 normal human skin fibroblast cell line was cultured in Eagle’s Minimum Essential Medium with Earle’s salts and non-essential amino acids supplemented with 15% fetal bovine serum (Biochrom, Berlin, Germany) and 2.2 g/l sodium bicarbonate anhydrous. Fibroblasts were seeded at 5 × 10⁴ cells/100 μl culture medium per well in black 96-well flat-bottom plates and incubated at 37°C in 5% CO₂ for 24 h to allow attachment. Cells were separately treated with either 8 μg/ml DDC, 32 μg/ml Cu²⁺ or DDC-Cu²⁺ (8 μg/ml DDC + 32 μg/ml Cu²⁺) for 18 h. The effect of the compounds on fibroblast viability was assessed with the CellTiter-Glo^® Luminescent Viability Assay (Promega Corporation, WI, United States) according to the manufacturer’s instructions and luminescence was measured on a FLUOstar OPTIMA plate reader. Equation 3 was used to quantify the percentage of fibroblast viability, where the luminescence intensity of treated and untreated fibroblast cells is represented by I_treatment and I_untreated, respectively, and I_blank represents the background luminescence of the CellTiter-Glo® reagent.

Cell viability assays were performed by seeding 5,000 cells/well in a 96-well opaque walled plate. Replicates were then treated with the BRAF inhibitor, dabrafenib (Sellekchem S2807), MEK inhibitor, trametinib (Selleckchem S2673), or combined treatment. The CellTiter-Glo Luminescent Viability Assay (Promega G7571) was used to measure cell viability at 24, 48, and 72 hours following the manufacturer’s protocol. Luminescence was measured using the Tecan M1000 Pro Microplate Reader.

RA FLS were cultured in 96-well plates (2 x 10³ cells/well) in DMEM, 5% FBS, 1% glutamine and 1% penicillin/streptomycin. Cells were treated with 400 ng/ml of rWnt5a for 24, 48, 72, and 96 h and proliferation was determined with the CellTiter-Glo luminescent viability assay (Promega, Wisconsin, USA) following the manufacturer’s instructions.

Single-cell suspensions of PDX cells were plated in 96-well plates at 16,000 cells per well in M87 medium and treated with 516 drugs (ApexBio DiscoveryProbe FDA-approved Drug Library) at 10 µM^{22 (link)}. After 72 h, the CellTiter-Glo Luminescent Viability Assay (Promega) was used according to the manufacturer’s protocol. Cell viability was quantified by normalizing treated wells to vehicle (0.1% DMSO) wells to produce a percent of vehicle value. Drug cytotoxicity was compared between the parental WHIM2 and erlotinib-resistant WHIM2 PDXs. Three separate tumors were tested in duplicate, and replicates were then averaged for each PDX.