Culture supernatants (10 μl) were transferred into a black 96-well plate (Costar). The adenylate kinase detection reagent (50 μl) (ToxiLight BioAssay, Lonza) was added to each well and the plate was incubated for 5 min at room temperature. Luminescence was measured in a microplate luminometer (Applied Biosystems). The results were expressed as relative luminescent unit (RLU).
Toxilight bioassay
Manufactured by Lonza
Sourced in Switzerland, Germany
The ToxiLight bioassay is a luminescent-based cell cytotoxicity assay used to measure the release of adenylate kinase from damaged cells. It provides a quantitative measure of cell membrane integrity and cell death.
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Lab products found in correlation
Triton x 100, by Merck Group (4 mentions)
Polarstar omega, by BMG Labtech (3 mentions)
Mithras lb940 microplate reader, by Berthold Technologies (3 mentions)
Caspase glo 3 7 assay, by Promega (3 mentions)
384 well plate, by PerkinElmer (2 mentions)
Half black 96 well plate, by Corning (2 mentions)
Black 96 well plate, by Corning (1 mentions)
Microplate luminometer, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Probenecid, by Merck Group (1 mentions)
Mithras lb 940, by Berthold Technologies (1 mentions)
Cell proliferation kit 2 xtt, by Roche (1 mentions)
Mithras lb 940 luminometer, by Berthold Technologies (1 mentions)
21 protocols using toxilight bioassay
1
Adenylate Kinase Activity Assay
2
Cell Viability and Cytotoxicity Screening
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Vero and HEp-2 cells were screened for cellular viability and cytotoxicity following siRNA transfection by CellTiter96 Non-Radioactive Cell Proliferation Assay Kit and ToxiLight bioassay (Lonza, Walkersville, MD) following manufacturer’s instructions. Cellular toxicity was evaluated at 0 and 48 h post-transfection. Data was normalized to siTOX control (100% cytotoxicity). siRNAs which resulted in >10% toxicity were excluded from the screen. Cell growth assessment revealed no significant differences in cell adherence or doubling time between KD and wild type cells.
3
Bioluminescent Cytotoxicity Evaluation
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The bioluminescent ToxiLight bioassay (Lonza) is a cytotoxicity highly sensitive assay designed to measure cell membrane damage. After 24 h of treatments, 5 µl of the clear fluid above a sediment was deposited in a 384-well plate (Perkin Elmer). Then 20 μl of the Adenylate Kinase Detection Reagent (AKDR) were added to each well and the plates were shaken. As a positive control for lysis 10 % Triton X-100 (Sigma-Aldrich) in medium was used, the negative control was medium alone. After 5-min incubation of the supernatant with the AKDR, the luminescence was measured in a plate reader (POLARstar Omega, BMG Labtech). The results were expressed as percent of positive control, which corresponds to the percentage of dead cells with respect to the control sample.
4
Bioluminescent Cytotoxicity Assay Protocol
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The bioluminescent ToxiLight bioassay (Lonza) is a highly sensitive cytotoxicity assay designed to measure cell membrane damage. After incubation of cells with the tested compounds, 10 μl of cell supernatant was deposited in a new 96-well plate. Then, 40 μl of the Adenylate Kinase Detection Reagent (AKDR) was added per well. After 5 minutes of incubation at 22°C, the luminescence intensity was measured in a multifunction plate reader (POLARstar Omega, BMG Labtech).
5
Adenylate Kinase Cytotoxicity Assay
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10 μl samples of culture supernatant were transferred on a half black 96 well plate (Costar). To each well, 50 μl of the adenylate kinase detection reagent (ToxiLight® BioAssay, Lonza) was added and the plate was incubated for 10 min at room temperature. Luminescence was measured in a Mithras LB940 Microplate Reader (Berthold Technologies). The results were expressed as relative luminescent unit (RLU).
6
Probenecid Antiviral Efficacy Assay
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A working stock of probenecid (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Sigma) and dilutions of the working stock were resuspended in PBS (Gibco). Cellular toxicity was determined using a ToxiLight Bioassay (Lonza). Vero E6 cells, HEp-2 cells, or undifferentiated NHBE cells were plated overnight at 104 cells/well in 96-well flat-bottom plates (Costar). Cells were either pretreated for 24h prior to infection (prophylactically) or therapeutically at 24h post-infection with probenecid at different concentrations, i.e., 100, 50, 25, 12, 6, 3, 1, 0.5, 0.2, 0.1, 0.05, 0.01, or 0 µM. Subsequently, the media and probenecid were removed and the cells were infected with RSV A2, RSV B1, or Memphis-37 at MOI = 0.1. At 72 h post-infection the plates containing the cells were frozen at −80 °C the freeze-thawed 3X and the cell-free supernatants were used for log10 dilutions in RSV plaque assays.
7
Measuring Cell Death via Adenylate Kinase
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To measure the adenylate kinase (AK) activity in culture supernatants as a marker of cell death, the ToxiLight® BioAssay (Lonza, Norwest, Australia) was utilized. Briefly, culture supernatant samples (10 μL) were transferred onto a black 96-well half-area plate (Costar, Washington, DC, USA). A detection reagent (50 μL) was added to each well, and the plate was incubated for 10 min at room temperature. Luminescence was measured using a Mithras LB940 Microplate Reader (Berthold Technologies, Bad Wildbad, Germany). The results were expressed as relative luminescent units (RLU).
8
Cytotoxicity Assay Using Bioluminescent ToxiLight
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The bioluminescent ToxiLight bioassay (Lonza) is a cytotoxicity highly sensitive assay designed to measure cell membrane damage. It quantitatively measures the release of Adenylate Kinase (AK) from the membranes of damaged cells. AK is a protein presented in all eukaryotic cells, which is released into the culture medium when cells die. The enzyme actively phosphorylates ADP and the resultant ATP is then measured using the bioluminescent firefly luciferase reaction with the ToxiLight reagent. The emitted light intensity expressed as a RLU value is linearly related to the adenylate kinase activity. After 24h of treatments, 5 µl of the clear fluid above sediment was transferred into 384-well plate (Perkin Elmer). Then 20 μl of the Adenylate Kinase Detection Reagent (AKDR) was added. As a positive control for lysis 10% Triton X- 100 (Sigma-Aldrich) in growth medium is used, the negative control is growth medium alone. The luminescence was measured in a plate reader (POLARstar Omega, BMG Labtech) after 5 minutes of incubation. The results were expressed as a percentage of positive control, which corresponded to the percentage of dead cells with respect to the control sample.
9
Spraying Effects on Cell Health
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To evaluate the influence of spraying on cell health, assays for apoptosis (Caspase-Glo 3/7 assay, Promega, Walldorf, Germany) and necrosis (ToxiLight BioAssay, Lonza, Cologne, Germany) were performed, according to the manufacturers’ instructions. Based on previous results, all assays were carried out with cells sprayed with the following spray parameters: high flow rate and a working distance of 9 cm. Cells (n = 3) were sprayed at a concentration of 2 × 106 cells/mL, diluted with EGM2 medium and seeded on RGD-coated polydimethylsiloxane (PDMS) membranes for further static or dynamic cultivation.
10
Optimized hNPC Culture and Antiviral Evaluation
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hNPCs were cultured under orbital rotation for three days to achieve optimal growth. One hour prior to infection, porcine intestine mucosal heparin (Celsus), heparin derivatives, and fractions were added at a final concentration of 100 µg/mL. The NS were then infected with the Brazilian 2016-INMI-1 isolate at a MOI of 1. Viral supernatants were collected at 3 and 6 days post-infection. To assess cell death, adenylate kinase activity levels were measured in the supernatants using the ToxiLight Bioassay from Lonza. Additionally, the viral titers were determined by a PFA.
hNPCs grown in 2D were initially seeded at a density of 2.5 × 105 cells/well in 24-well flat-bottom plastic plates using 500 μL of complete medium. After 24 h, hNPCs were subjected to pre-treatment with either Sofosbuvir (AstaTech, Inc., Bristol, PA, USA) or heparin and a combination of both. Subsequently, the cells were inoculated with the PRVABC59 isolate at a MOI of 1. Following a 6-day incubation period, viral supernatants were collected. The viral titers were determined using PFA.
hNPCs grown in 2D were initially seeded at a density of 2.5 × 105 cells/well in 24-well flat-bottom plastic plates using 500 μL of complete medium. After 24 h, hNPCs were subjected to pre-treatment with either Sofosbuvir (AstaTech, Inc., Bristol, PA, USA) or heparin and a combination of both. Subsequently, the cells were inoculated with the PRVABC59 isolate at a MOI of 1. Following a 6-day incubation period, viral supernatants were collected. The viral titers were determined using PFA.
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