Celltiter glo 2

Cell viability was assessed using the CellTiter-Glo 2.0 assay (Promega) according to the manufacturer's instructions. Briefly, cells were plated at a density of 3.3 × 10⁴ cells per well in a 96 well plate and incubated overnight (37 °C, 5% CO₂). Cells were treated with increasing concentrations of inhibitor and incubated in lipoprotein depleted serum for 72 h (37 °C, 5% CO₂). The plate was equilibrated to room temperature for 30 min prior to the addition an equal volume of CellTiter-Glo 2.0 (Promega) reagent to media. The contents were mixed for 2 min on an orbital shaker to induce cell lysis and the plate incubated at room temperature for 10 min prior to the detection of a luminescent signal.

Flp-In BRD4 WT or 4A cells were treated with 0.5 μM indole-3-acetic acid (IAA) and 50 ng/mL Dox for 1 day before incubation with DMSO control or different concentration of (+)-JQ1. After 3 days, cell viability assays were performed using CellTiter-GLO 2.0 (Promega, Madison, WI, USA) according to the manufacturer’s instruction. Relative viability was calculated by normalization to the DMSO control. The corresponding dose-inhibition curves were analyzed using nonlinear regression method, log(inhibitor) vs. response-Variable slope function in the GraphPad Prism 5.0 software (San Diego, CA, USA).
For the drug combination assays, MDA-MB-231 cells were treated with DMSO or indicated doses of (+)-JQ1 and/or RO-3306, individually or in combination; Flp-In BRD4 WT or 4A cells were treated with 0.5 μM IAA and 50 ng/mL Dox for 1 day before incubation with DMSO control or indicated doses of (+)-JQ1 and/or RO-3306 individually or in combination. 3 days after drug treatment, cell viability assays were performed using CellTiter-GLO 2.0 (Promega) according to the manufacturer’s instruction. Relative cell viability was determined by normalizing the drug treatment values to the DMSO control values. CI values were analyzed with CompuSyn software [67 (link)].

The cell lines StromaNKtert (CVCL_4667), HS-5 (CRL-11882), WI-38 (ATCC#CCL-75), MEC-1 (CVCL_1870) were cultivated at 37°C and 5% CO₂ in RPMI 1640 containing 10% FBS, supplemented with GlutaMAX (2 mM), 100 U/ml penicillin and 100 μg/ml streptomycin. The following additional reagents were used: PE annexin V (Cat# 640907, BioLegend), rabbit anti-human CD248 (AB67273, Abcam, Berlin, Germany), anti-β-actin antibody (Santa Cruz), Vybrant-DiD (red, 40 nM 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindo-dicarbo-cyanine, 4-chlorobenzenesulfonate salt, Thermo Fisher Scientific, Munich, Germany), CFSE (green, 6-carboxyfluorescein succinimidyl ester, Thermo Fisher Scientific, Munich, Germany), CellTiter-Glo 2.0 (Promega, Walldorf, Germany).

The CellTiter Glo^® 2.0 and CellTiter Glo^® 3D cell viability assay systems (Promega, Madison, WI, USA) were used to quantify cytotoxicity by measuring metabolic active cells through ATP presence through a luminescent luciferase reaction that is directly proportional to viable cells in the culture. Briefly, target cells and effector cells were co-cultured in the indicated E:T ratios, cell numbers, treatments, and time points. At the experimental end point, the viability assay reagent was added to the co culture in a 1:1 ratio (total culture volume–assay reagent) followed by shaking of the assay plate, incubation for 10 min (2D) or 25 min (3D) at room temperature, and subsequently measured using a Tristar 3 multimode plate reader (Berthold Technologies, Bad Wildbad, Germany). Target cell lysis was quantified using the formula: $$\mathrm{Cytotoxicity} (\%)=100-(\frac{Sample release-Effector cell release}{Maximum release})\times 100$$

The migration potential of the transduced NK cell lines was determined utilizing cell culture inserts (ThinCert^®; Greiner Bio-One, Frickenhausen, Germany) with an 8 µm pore size. A fixed number of 50,000 cells were loaded in the cell culture inserts at a total volume of 100 μL assay medium without chemokines. The inserts were placed in 24-well dishes containing 0.5 µg/mL of the respective chemokine (CCL17/CCL22 (Immunotools)) diluted in 500 μL assay medium. After 4 h of incubation at 37 °C and 5% CO₂, migrated cells were quantified by measuring ATP using CellTiter Glo^® 2.0 (Promega) following the manufacturer’s protocol.

The cell viability assay CellTiter-Glo 2.0 (Promega, Mannheim, Germany) was used in EGC after treatment with cytokines as described by the manufacturer. In brief, EGC were grown to confluence on 96-well plates. After serum starvation, cells were incubated with TNFα, LPS and LTA for 24 h. Staurosporine (Sigma Aldrich, St. Louis, USA) was used at a concentration of 500 ng/mL. Luminescent measurements were performed by using a microplate reader (Tecan Microplate Reader, MTX Lab systems, Bradenton, USA).

Primary cells were stimulated for 2 hours for MSD experiments, for 4 hours for qRT-PCR experiments, and for 20 hours for cell viability and protein secretion readouts by CellTiter-Glo and ELISA, respectively. For OPC-related conditioned media cells were treated in serum-free OPC media for 24 hours and conditioned media was applied to OPCs for 72 hours. Typically, primary cells were pre-treated with TAK1 (5z-7, 100nM), cIAP1/2 (Smac mimetic, 1 μM), pan-caspase (zVAD, 20 μM), and RIPK1 kinase inhibitors (Nec-1 s, 30 μM)for 1 hour prior to stimulation with recombinant murine or human TNF (50ng/mL). After 20 hours, cell viability was assessed via CellTiter-Glo 2.0 (Promega #G9243; according to manufacturer’s instructions) and normalized to DMSO.