The TNBC MDA-MB-231 cells (1.5 × 104/cm2) were plated and treated in complete medium and incubated for 24 h, as described. Cytotoxicity was measured with a Promega CytoTox-Glo® assay kit, following the manufacturer’s recommendations. Briefly, the cytotoxicity evaluation was based on the measurement of the fluorogenic cell-impermeant peptide substrate, generated by the released dead-cell protease due to membrane integrity loss. The fluorescent signal was measured using an EnVision™ microplate reader.
Cytotox glo assay kit
Manufactured by Promega
Sourced in United States
The CytoTox-Glo assay kit is a luminescent-based cell viability and cytotoxicity assay. It measures the release of a specific protease from damaged cells as an indicator of cytotoxicity.
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4 protocols using cytotox glo assay kit
1
TNBC Cell Cytotoxicity Evaluation
2
Cytotoxicity Evaluation of Gentian Violet
3
Cytotoxicity Evaluation using CytoTox-Glo
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The cytotoxicity was determined by using the CytoTox-Glo assay kit from Promega Corporation (Fitchburg, WI, USA) according to Niles et al35 (link) and manufacturer’s protocol. Shortly, 2.5×105 cells were seeded in black µClear 96-well plates (Greiner Bio-One), and incubated overnight prior to adding controls and samples to the cells. The exposure time to the substances was 4 hours. Digitonin (50 µg/mL) was used as cytotoxicity standard (positive control), cell culture medium as negative control, and water to exclude toxic effects of the solvent. The measured data were blank-corrected and normalized to the negative control.
4
Assessing Cell State Changes via Luminescent Assays
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5000 cells per reaction were applied to detect apoptosis, senescence and necrosis. All reactions were measured using a luminometer (Glo Max Multi + Detection System; Promega).
Senescence was analyzed using the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Wisconsin, USA). 10 μl of Digitonin (30 μg/ml), added to the cells in a separate well 15 min before cell lysis, served as positive control to measure a decrease of cellular viability of 100%.
Necrosis was analysed using the CytoTox-Glo® Assay kit (Promega). Ionomycin (Selleckchem) was used for positive control. Two hours before measurement, 50 μl of Ionomycin (100 μM), was added to the cells in a separate well. After adding 50 μl of the AAF-Glo® reagent to each well, cells were incubated for 15 min at room temperature, protected from light.
The apoptotic potential of the cells was analysed using the Caspase-Glo® 3/7 Assay (G8093, Promega). 100 μl of required cells/well were placed into a white 96-well plate. Staurosporine (10 μM, Selleckchem) served as positive control and was given to the cells in a separate well 4 h before measurement. After adding 100 μl of Caspase-Glo® reagent to each well, cells were incubated for 30 min at room temperature.
Changes in cell state were calculated as percentage of signal gained by the positive control normalized to the baseline (untreated cells).
Senescence was analyzed using the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Wisconsin, USA). 10 μl of Digitonin (30 μg/ml), added to the cells in a separate well 15 min before cell lysis, served as positive control to measure a decrease of cellular viability of 100%.
Necrosis was analysed using the CytoTox-Glo® Assay kit (Promega). Ionomycin (Selleckchem) was used for positive control. Two hours before measurement, 50 μl of Ionomycin (100 μM), was added to the cells in a separate well. After adding 50 μl of the AAF-Glo® reagent to each well, cells were incubated for 15 min at room temperature, protected from light.
The apoptotic potential of the cells was analysed using the Caspase-Glo® 3/7 Assay (G8093, Promega). 100 μl of required cells/well were placed into a white 96-well plate. Staurosporine (10 μM, Selleckchem) served as positive control and was given to the cells in a separate well 4 h before measurement. After adding 100 μl of Caspase-Glo® reagent to each well, cells were incubated for 30 min at room temperature.
Changes in cell state were calculated as percentage of signal gained by the positive control normalized to the baseline (untreated cells).
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