Plasma membrane integrity was assessed by the detection of adenylate kinase (AK) release into supernatant of the incubations. AK was determined with the ToxiLight assay kit (Lonza, Basel, Switzerland) and as described previously (Felser et al., 2013 (link)).
Toxilight assay kit
Manufactured by Lonza
Sourced in Switzerland
The ToxiLight assay kit is a bioluminescent-based kit designed to detect the release of adenylate kinase (AK) from damaged cells. It provides a quantitative measurement of cytotoxicity.
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Lab products found in correlation
Fluostar omega, by BMG Labtech (2 mentions)
Victor x5 plate reader, by PerkinElmer (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Amplification grade dnase 1 kit, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
2 propanol, by Merck Group (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Human il 1β, by R&D Systems (1 mentions)
Tnf α duoset elisa kits, by R&D Systems (1 mentions)
Nuclease free water, by Qiagen (1 mentions)
Primers for qpcr, by Eurofins (1 mentions)
6 protocols using toxilight assay kit
1
Plasma Membrane Integrity Evaluation
2
Assessing Cell Viability via AK Levels
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Cell viability was assessed by measuring adenylate kinase (AK) levels using ToxiLight™ assay kit (Lonza). Briefly, HepG2/C3A flat cells and spheroids supernatants were collected in duplicate and 20 µL was transferred to a 96-well white-walled plate (flat bottom clear). For each assay plate, a standard curve was prepared using different amounts of cells (156 to 10,000 HepG2/C3A cells per well) lysed in digitonin lysis buffer (Promega). One hundred microliters of adenylate kinase detection reagent (Lonza) was added to each well and the content was homogenized by gently pipetting up and down. Bubbles in the assay were removed by centrifugation with pressure. The plate was incubated for 20 min at room temperature and read in a Victor X5 plate reader (Perkin Elmer), in luminescent mode.
3
Cell Culture and Cytokine Analysis Protocol
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RPMI-1640 media, 2-mercaptoethanol (ME), Hank’s Balanced Salt Solution containing MgCl2 and CaCl2 (HBSS), sodium pyruvate, and TRIzol® reagent were purchased from Thermo Fisher Scientific. Fetal calf serum (FCS), bovine serum albumin (BSA), Penicillin/Streptomycin (P/S), D-glucose, trypsin, accutase, phorbol 12-myristate-13-acetate (PMA), LPS, endotoxin-free H2O, phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-propanol, and the amplification grade DNase I Kit were purchased from Sigma-Aldrich/Merck. The ToxiLight assay kit was purchased from Lonza. Ethanol and sulfuric acid were purchased from Roth. Nuclease-free water was purchased from Qiagen. The iScript™ cDNA Synthesis Kit and the iQ™ SYBR® Green Supermix were purchased from Bio-Rad. Primers for qPCR were purchased from Eurofins. The Human IL-1β, IL-6, IL-8, and TNFα DuoSet ELISA Kits were purchased from R&D Systems.
4
Cytotoxicity of Doxorubicin Nanoformulations
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The cytotoxicity induced by the exposure of HepG2 and A549 cells to different concentrations of free DXR, NS-DXR, or plain NS (identical concentration of nanospheres as NS-DXR, but without DXR) was determined using the ToxiLight assay kit (Lonza Bioscience, Basel, Switzerland). The assay uses luminescent detection to measure the levels of adenylate kinase (AK) released by the damaged cells into the culture media.
HepG2 and A459 cells were seeded in a 96-well culture plates at a density of 104 cells/well and after 48 h the cells were treated with various concentrations of NS or NS-DXR (3.9 ÷ 125 μg/mL) and free DXR, at the corresponding concentrations as entrapped into NS-DXR (0.078 ÷ 2.5 μg/mL) for 24 h. Then, the culture medium was collected, and the cytotoxicity was assessed using the bioluminescent kit according to the manufacturer’s instructions. The results were expressed as fold change relative to the results obtained for the untreated control cells.
HepG2 and A459 cells were seeded in a 96-well culture plates at a density of 104 cells/well and after 48 h the cells were treated with various concentrations of NS or NS-DXR (3.9 ÷ 125 μg/mL) and free DXR, at the corresponding concentrations as entrapped into NS-DXR (0.078 ÷ 2.5 μg/mL) for 24 h. Then, the culture medium was collected, and the cytotoxicity was assessed using the bioluminescent kit according to the manufacturer’s instructions. The results were expressed as fold change relative to the results obtained for the untreated control cells.
5
Cytotoxicity Evaluation in 3D Spheroids
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Adenylate kinase (AK) was measured in the growth medium (140 µl) of each experimental group in the 3D spheroid containing bioreactors to determine cytotoxicity, at time points 0, 4, 8, 12 and 24 h following each medium exchange,
A c c e p t e d M a n u s c r i p t
for a duration of 96 h, using the Lonza Toxilight assay kit (Cat No LT07-117).
Growth medium was placed in microtiter plates in triplicate and diluted with five volumes of AK detection reagent. The plate was then incubated in the dark for 20 min before measuring luminescence using a FluoStar Omega ® (BMG Labtech, Ortenberg, Germany). A standard curve was prepared in the same way for each assay plate, using a calibrated cellular lysate as standard (4.29 million C3A cells/ml lysed in lysis buffer). Gain was adjusted based on the highest standard curve values.
A c c e p t e d M a n u s c r i p t
for a duration of 96 h, using the Lonza Toxilight assay kit (Cat No LT07-117).
Growth medium was placed in microtiter plates in triplicate and diluted with five volumes of AK detection reagent. The plate was then incubated in the dark for 20 min before measuring luminescence using a FluoStar Omega ® (BMG Labtech, Ortenberg, Germany). A standard curve was prepared in the same way for each assay plate, using a calibrated cellular lysate as standard (4.29 million C3A cells/ml lysed in lysis buffer). Gain was adjusted based on the highest standard curve values.
6
Cytotoxicity Assay for Cell Death
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AK content was measured in the growth medium (140 µl) of each experimental group to determine cell death in 24 h intervals for the duration of the 21-day study, using the Lonza Toxilight assay kit.
Growth medium was placed in microtitre plates in triplicate and diluted with five volumes of AK detection reagent. The plate was then incubated in the dark for 20 min before measuring
luminescence using a FluoStar Omega ® (BMG Labtech). A standard curve was prepared in the same way for each assay plate using a dead-cell standard (4.29 x 10 6 HepG2/C3A cells per ml, which were lysed in lysis buffer). Gain was adjusted based on the highest standard curve values.
Growth medium was placed in microtitre plates in triplicate and diluted with five volumes of AK detection reagent. The plate was then incubated in the dark for 20 min before measuring
luminescence using a FluoStar Omega ® (BMG Labtech). A standard curve was prepared in the same way for each assay plate using a dead-cell standard (4.29 x 10 6 HepG2/C3A cells per ml, which were lysed in lysis buffer). Gain was adjusted based on the highest standard curve values.
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