The cytotoxic effect of molecules containing the type C protease were compared in differentiated neuroblastoma using the Deep Blue Cell Viability Kit (BioLegend). Following treatment of differentiated Nanoluc-VAMP2 expressing SiMa neuroblastoma with botulinum constructs for 65 h in 96 well plates, 15 μL of Deep Blue Cell Viability reagent was added to each well before incubation at 37 °C for 6 h. Fluorescence of the metabolised reagent was measured at Excitation 560 nm and Emission 590 nm. Wells containing differentiation media alone were used as a negative control. Cell viability was normalised using the mean average of untreated cells as the 100% value.
Deep blue cell viability kit
Manufactured by BioLegend
Sourced in United States, United Kingdom
The Deep Blue Cell Viability Kit is a fluorescence-based assay designed to quantify the number of viable cells in a population. It utilizes a proprietary dye that selectively stains living cells, allowing for accurate determination of cell viability.
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Lab products found in correlation
Infinite m1000 pro, by Tecan (1 mentions)
Calcein am, by BioLegend (1 mentions)
Ldh cytox assay kit, by BioLegend (1 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (1 mentions)
Celltiter glo 2, by Promega (1 mentions)
Birinapant, by Apexbio (1 mentions)
Necrostatin 1 nec 1, by Selleck Chemicals (1 mentions)
Z val ala dl asp ome fluoromethylketone z vad, by Bachem (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Dmi6000b inverted microscope, by Leica (1 mentions)
Synergy 2 multi mode, by Agilent Technologies (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
13 protocols using deep blue cell viability kit
1
Cytotoxicity of Type C Protease Molecules
2
Viability and Cytotoxicity Assay of HDM-Treated Cells
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Cells were seeded in black 96-well plates with clear bottoms (Greiner, Frickenhausen, Germany) at a cell density of 5,000 cells per well in 200 μl DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 2 mM GlutaMAX. After 24 h, cells were stimulated with house dust mite (HDM) from Greer Laboratories (catalog no. XPB70D3A2.5; Lenoir, NC).
Cell viability was determined after 20 h with the Deep Blue Cell Viability Kit (BioLegend, San Diego, CA). The reduction of resazurin to resorufin was measured after 4 h incubation with a TECAN Infinite M1000 Pro fluorescence plate reader (Männedorf, Switzerland) at excitation and emission wavelengths of 550 nm and 610 nm, respectively.
For live/dead cell assays, 24 h after treatments, plates were centrifuged at 300 g for 5 min at room temperature, and 100 μl of the media was removed and replaced by 100 μl of HBSS containing 3 μM calcein-AM (BioLegend, San Diego, CA) and 5 μM propidium iodide (PI). After 30 min of incubation at 37°C, PI fluorescence was measured with a TECAN Infinite M1000 Pro fluorescence plate reader at 530 nm/620 nm. Plates were washed and refilled with 200 μl of HBSS per well, and calcein fluorescence was measured at 485 nm/535 nm.
Cell viability was determined after 20 h with the Deep Blue Cell Viability Kit (BioLegend, San Diego, CA). The reduction of resazurin to resorufin was measured after 4 h incubation with a TECAN Infinite M1000 Pro fluorescence plate reader (Männedorf, Switzerland) at excitation and emission wavelengths of 550 nm and 610 nm, respectively.
For live/dead cell assays, 24 h after treatments, plates were centrifuged at 300 g for 5 min at room temperature, and 100 μl of the media was removed and replaced by 100 μl of HBSS containing 3 μM calcein-AM (BioLegend, San Diego, CA) and 5 μM propidium iodide (PI). After 30 min of incubation at 37°C, PI fluorescence was measured with a TECAN Infinite M1000 Pro fluorescence plate reader at 530 nm/620 nm. Plates were washed and refilled with 200 μl of HBSS per well, and calcein fluorescence was measured at 485 nm/535 nm.
3
Evaluating Combinatorial Cytotoxicity in Cells
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Cell viability was detected using the Deep Blue Cell Viability Kit (BioLegend, San Diego, CA, USA) and determined relative to the viability of the control cells (100%) ± standard deviation in three independent experiments. Cytotoxic dose (CD50, the dose at which 50% of cells die) of drugs and the combination index (CI) of drug combination were evaluated using Compusyn software v1.0 (ComboSyn Incorporated, Paramus, NJ, USA).
To assess the cytotoxicity of an individual drug, each reagent (TMZ, VCR, CCNU, and PCB) was added to cells at the concentrations described previously.
To analyze the combined effect of the drugs, VV-GMCSF-Lact and TMZ were added at different time intervals as needed. Concentrations of VV-GMCSF-Lact were equal to CD50 for each cell culture. For the test, concentrations of TMZ were chosen to be less than CD50, CD50, and more than CD50 for each culture. TMZ was added to cells after 12, 24, 36, 48, or 60 h after virus administration. The total time of cell incubation with the drugs was 72 h at 37 °C in an atmosphere of 5% CO2.
After incubation with drugs, commercial reagent was added into wells, and the optical density was measured on the spectrophotometer Apollo LB 912 (Berthold Technologies GmbH & Co., KG, Bad Wilbad, Germany) according to the manufacturer’s instructions.
To assess the cytotoxicity of an individual drug, each reagent (TMZ, VCR, CCNU, and PCB) was added to cells at the concentrations described previously.
To analyze the combined effect of the drugs, VV-GMCSF-Lact and TMZ were added at different time intervals as needed. Concentrations of VV-GMCSF-Lact were equal to CD50 for each cell culture. For the test, concentrations of TMZ were chosen to be less than CD50, CD50, and more than CD50 for each culture. TMZ was added to cells after 12, 24, 36, 48, or 60 h after virus administration. The total time of cell incubation with the drugs was 72 h at 37 °C in an atmosphere of 5% CO2.
After incubation with drugs, commercial reagent was added into wells, and the optical density was measured on the spectrophotometer Apollo LB 912 (Berthold Technologies GmbH & Co., KG, Bad Wilbad, Germany) according to the manufacturer’s instructions.
4
Cell Viability Assay for Multiple Myeloma
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For viability assays, mouse or human MM cells were seeded in 96-well black culture plates and treated with different drugs for 48 h. Cell viability was quantified using a Deep Blue Cell Viability Kit (BioLegend) and analyzed with a Skanit Varioskan Flash 2.4.3 (Thermo Scientific) fluorometer. Treatments were administered to cells at a density of 0.3 × 106 cells per ml, and all tests were performed in triplicate. After treatment, cells were subjected to RT–qPCR or western blot analyses, as indicated, according to previously reported methods70 (link).
5
Cytotoxicity Assays for Losartan and sKlotho
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To investigate if treatment with losartan or sKlotho induced cytotoxicity, LDH-Cytox Assay Kit (BioLegend) was used following the “homogeneous assay using viable cells” protocol for LDH measurement. Additionally, the Deep Blue Cell Viability kit (BioLegend) was used as a secondary measure of cytotoxicity induced by losartan or sKlotho treatment. For both assays, cells were cultured in a 96-well plate for 24 h, serum starved overnight, and treated with control (Water), DMSO, losartan, or sKlotho in 0.5% serum media for 24 h. For shRNA-transduced cells, an equal number of NT-shRNA or AT1-shRNA or AT1-shRNA-2 cells were cultured in a 96-well plate for 24 h, serum starved overnight, and media replaced with 0.5% serum media for additional 24 h. The measurements (luminescence or fluorescence readings) were taken and analyzed according to the manufacturers’ protocols. For LDH assay, water-treated samples were used as “low control” for percent cytotoxicity calculation. Comparisons were made between DMSO and losartan or water and sKlotho. For shRNA-transduced cells LDH assay, NT-shRNA samples were used as “low control” for percent cytotoxicity calculation. For viability assay, fluorescence levels for losartan, sKlotho, or AT1-shRNA/AT1-shRNA-2 were determined relative to DMSO, water, or NT-shRNA respectively.
6
Cell Viability Assay for VV-GMCSF-Lact
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Cell viability was detected using the Deep Blue Cell Viability Kit (BioLegend, San Diego, CA, USA). The cells that had reached 60% confluence in a 96-well plate were incubated for 72 h with VV-GMCSF-Lact; the multiplicity of infection ranged from 0.0012 to 10 PFU per cell. After incubation, commercial reagent was added into wells, and the optical density was measured according to the manufacturer’s instructions. Cell viability was determined relative to the viability of the control cells’ (100%) ± standard deviation in three independent experiments.
7
Cell Metabolic Activity Assay
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Resazurin assay (Deep Blue Cell Viability™ Kit, BioLegend) was used in this study to determine cell metabolic activity after 10 days of direct ES or ES media treatment. The stock solution was diluted in GM to make 10% working solution. To implement the assay, cell culture supernatant was replaced with 1 ml of working solution before being incubated at 37°C for 1 h. Fluorescence intensity was measurement at excitation wavelength of 544 nm and emission wavelength of 590 nm. The measured intensity was subtracted by background readings.
8
Quantitative Cell Viability Assay Protocol
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Cells were seeded for viability assays on 96-well plates, ensuring that cells were dissociated into clumps of ∼10–25 cells in size prior to seeding. For gamma irradiation, cells were seeded into 12.5-cm2 flasks. Cells were treated at 5%–10% confluency with a range of concentrations (≥6) for each agent, in order to establish dose-response curves spanning 0%–100% cytotoxicity, where possible, with each treatment condition tested in ≥ 3 technical replicates. Following the appropriate treatment duration, cells were maintained in growth medium to allow cell division and processing of DNA damage. At 72 hr after treatment initiation, cell viability was quantified using the Deep Blue Cell Viability Kit (Biolegend), which measures the reduction of reazurin to fluorescent resorufin by viable cells. Fluorescence (Ex530/Em590) was measured using a plate reader. Data are presented as the amount of fluorescence of treated cells relative to that of control (media only or solvent-treated) cells and are representative of at least three independent experiments. IC50 values were calculated using Prism 7 software.
9
Assessing Cell Death Pathways
10
Resazurin-Based Cell Viability Assay
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Samples were incubated with 1 ml of 10% resazurin solution (Deep Blue™ Cell Viability Kit; BioLegend, UK) diluted in the complete media for 1 hr. The fluorescence intensity of the supernatant was measured by microplate reader at excitation wavelength of 544 nm and emission wavelength of 590 nm. The background readings were subsequently subtracted from the measured intensity before analysis. The measurement was carried out the day after the final stimulation or ES media treatment.
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