Celltiter glo ctg reagent

SH-SY5Y neuroblastoma cells purchased from ATCC (VA, USA) were grown, sub-cultured and treated with different concentrations (1, 5, 10, 20, 50, 100 μM) of pre-aggregated Aβ42 with or without the highest concentration of API that was not cytotoxic [Gandbhir and Sundaram et al., 2019 (link)]. Cell cultures were incubated for 72 hrs. and cell viability was assessed using CellTiterGlo (CTG) reagent from Promega. The cell growth inhibition from the cells that are exposed only to pre-aggregated Aβ42 was used to estimate the concentration of Aβ42 that results in 50% inhibition (IC₅₀). The IC₅₀ for Aβ42 was derived by dividing the mean cell growth by the mean blanked control cell growth for each concentration, then multiplied by 100. The percent toxicity data was fitted for the 4-Parameter (4P) curve, with interpolation set to 50. The concentration of the Aβ42 that results in 50% inhibition was calculated from this curve. A second experiment was done where SH-SY5Y cells were incubated with Aβ42 at IC₅₀ and with different concentrations (1, 5, 10, 20, 50, 100, 200 μM) of API. Cell viability was measured, and IC₅₀ for ATC is estimated as described [Gandbhir and Sundaram et al., 2019 (link)].

Compound mediated cytotoxicity of A549 cells were determined using the of CellTiter-Glo® (CTG) reagent (Promega Corp.). A549 cells were grown on surface modified T175 cell culture flasks in DMEM with 10% FCS, streptomycin (100 μg/ml) and 100 U/ml penicillin G. At about 80% confluency, cells were washed, trypsinised, resuspended and counted in RPMI-1640 medium before seeding into white 384 well microtiter plates (20 μl) at 500 cells/well and incubated at 37 °C in the presence of 5% CO₂ and compounds for 24 h. Prior to addition of cells, to each well of a 384 well microtiter plate were added test compounds (200 nl of 1 mM solution in 100% v/v DMSO), positive controls (paclitaxel with final concentration of 10 μM and 1% v/v DMSO) yielding 100% inhibition and negative controls (200 nl of 100% v/v DMSO) yielding 0% inhibition using the Echo 550® Liquid Handler. This was followed by addition of 20 μl/well of CellTiter-Glo® (CTG) reagent (Promega Corp.) to each well and the luminescence signal detected using an EnVision® Multilabel 2103 Reader after 10 min incubation in the dark. The raw data were normalised relative to the positive and negative controls yielding the % inhibition for each compound.

HA- or Ova-expressing Cas9⁺ cancer cell lines were transduced with lentivirus bearing gRNAs targeting genes of interest or intergenic control regions. Transduced cells were selected with puromycin for around 72 h and seeded into 24- (25,000–50,000 cells per well) or 96- (1,500–5,000 cells per well) well plates in duplicates or triplicates. Following overnight incubation, cells were treated with preactivated CL4 or OT-1 CD8⁺ T cells at increasing effector-to-target ratios for around 48 h.
At the end point, CD8⁺ T cells and dead cancer cells were removed by gentle PBS wash, with cancer cell viability assessed by counting the remaining adherent cells in each well on a Coulter counter (24-well plates), via bioluminescence using a microtitre plate reader (96-well plate) or using CellTiter-Glo (CTG) reagent according to the manufacturer’s instructions (G7570, Promega). For selected experiments, cancer cell viability was also monitored by live-cell fluorescent microscopy using the IncuCyte. In all cases, cell viability relative to untreated control cells is shown.
For selected experiments, cancer cells were preincubated for 30 min with 100 μg/ml anti-TNF (MP6-XT22, Biolegend 506331) or anti-IFNγ (XMG1.2, Biolegend 505834) antibodies before addition of T cells to neutralize these cytokines during co-culture.