A terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assay was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche, U.S.A.), according to manufacturer’s instructions. Briefly, HSMM cell monolayers grown overnight on coverslips till 75% confluency were infected with CHIKV strain Singapore 072008 at an MOI of 10 for 2 hrs at 37 °C. Infected cells were subsequently fixed with 4% paraformaldehyde/PBS and permeabilised with 0.1% Triton-X/0.1% sodium citrate solution at daily time-points. Negative (without TdT) and positive (treated with 1 μM staurosporine for 2 hrs and harvested at 8 hrs post-infection [h.p.i.]) labeling controls were included, along with mock-infected controls. Samples were then subjected to labeling with the TUNEL reaction mixture prior to imaging analysis. Cell nuclei were counter-stained with DAPI. HSMM cells were infected with CHIKV strain Singapore 072008 at MOI of 10. At selected time intervals p.i., CHIKV-infected cells were processed in accordance with manufacturer’s instructions (Promega Caspase-Glo® 3/7 Assay kit) to determine caspase-3 activity. Luminescence of each sample well was analyzed in a plate-reading luminometer (GloMax® 96 Microplate, Promega). The activity of caspase 3 was expressed as relative luminescence unit (RLU) with respect to that of mock-infected cells.
Glomax 96 microplate
Manufactured by Promega
Sourced in United States
The GloMax 96 Microplate Luminometer is a compact and versatile instrument designed for precise measurement of luminescent signals in 96-well microplates. It utilizes a high-sensitivity photomultiplier tube (PMT) detector to accurately quantify light-based reporter assays, such as luciferase, aequorin, and ATP detection.
Automatically generated - may contain errors
Lab products found in correlation
Cck 8, by Dojindo Laboratories (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Caspase glo 3 7 assay kit, by Promega (1 mentions)
Muse oxidative stress kit, by Merck Group (1 mentions)
Celena s digital imaging system, by Logos Biosystems (1 mentions)
Glomax software, by Promega (1 mentions)
Beta glo assay system, by Promega (1 mentions)
Pierce 660 nm assay kit, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
9 protocols using glomax 96 microplate
1
Programmed Cell Death in CHIKV-Infected Cells
2
ROS Assay for Plant Immunity
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ROS assays were performed as described previously (Zhang et al., 2010 (link)). Leaf strips of 4-week-old plants were treated with 100 nM of flg22, 100 nM of elf18, or 0.1 mg/ml of chitin. Luminescence was detected with the GloMax 96 microplate luminometer (Promega).
3
Measuring Reactive Oxygen Species in Tomato
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The production of reactive oxygen species (ROS) was measured as described previously [53] (link). Eight 2 mm discs were excised from leaves of 3–4 week-old tomato plants grown in the greenhouse and luminescence was measured in a Glomax 96 microplate luminometer (Promega, Madison, WI, USA) at 2 min intervals for 20–50 min after the addition of the test solution.
4
MERS-CoV Pseudovirus Neutralization Assay
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MERS pseudovirus preparation and titration determination were performed as described15 (link). Briefly, the plasmids of 14 μg pCAGGS-MERS-S and 7 μg pNL4–3.luc.RE were cotransfected into 293T cells cultured in 100 mm dish. After 48 h, the supernatant containing pseudovirus was harvested, centrifuged and filtered through a 0.45 μΜ sterilized membrane. Single use aliquots (1.0 ml) were stored at 80 °C. The 50% tissue culture infectious dose (TCID50) was determined by infection of Huh7 cells39 (link).
For the neutralization assay, 100 TCID50/well pseudovirus was incubated with 2-fold serially diluted antibodies (4C2 and 4C2h from 48 ng/ml to 50 μg/ml, 2E6 from 12 ng/ml to 12.5 μg/ml) for 30 min at 37 °C. The mixtures were then used to infect Huh7 cells seeded in 96-well plates with 4 repeats. After 5 h incubation, the medium was replaced with DMEM containing 10% fetal bovine serum (FBS), and the samples were incubated for an additional 48 h at 37 °C. Luciferase activity was measured using a GloMax 96 Microplate luminometer (Promega). The 50% neutralization dose (ND50) was calculated using Prism.
For the neutralization assay, 100 TCID50/well pseudovirus was incubated with 2-fold serially diluted antibodies (4C2 and 4C2h from 48 ng/ml to 50 μg/ml, 2E6 from 12 ng/ml to 12.5 μg/ml) for 30 min at 37 °C. The mixtures were then used to infect Huh7 cells seeded in 96-well plates with 4 repeats. After 5 h incubation, the medium was replaced with DMEM containing 10% fetal bovine serum (FBS), and the samples were incubated for an additional 48 h at 37 °C. Luciferase activity was measured using a GloMax 96 Microplate luminometer (Promega). The 50% neutralization dose (ND50) was calculated using Prism.
5
Assessing Oxidative Stress in Keratinocytes
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The oxidation-sensitive dye, DCFDA, was used to determine the formation of intracellular ROS and NAC was used as a negative control. Briefly, HaCaT keratinocytes were seeded at a density of 1 × 104 cell/mL and treated with PS at the indicated concentrations (0–400 μg/mL) for 20 h followed by exposure with 1000 μM H2O2 for 4 h. The cells were washed with PBS and immediately treated with 10 μM DCFDA. Intracellular ROS generation was measured by a GloMax® 96 microplate fluorometer (Promega; Madison, WI, USA). In a parallel experiment, live imaging of HaCaT keratinocytes was detected by a CELENA® S digital imaging system (Logos Biosystems; Anyang, Korea). ROS− and ROS+ cell populations were determined by flow cytometry. Briefly, HaCaT keratinocytes were incubated with Muse® Oxidative Stress Kit (MCH100111, EMD Millipore) for 30 min. ROS− and ROS+ cell populations were measured by Muse® Cell Analyzer.
6
Organoid viability assay with drug treatments
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Mock-infected organoids were seeded in 30 μl BME drops in 48 well plates overlaid with 250 μl of DM in duplicate per condition. Organoids were mock-treated with DMSO, or treated with 10 μM PYR or K98. As a positive control, organoids were also treated with 10 μg/ml of puromycin. 6 days post seeding for the mock and drug-treated organoids and 9 days post seeding for two additional wells of mock-treated organoids, 120 μl of the supernatant was removed and 150 μl of Cell-Titre Glo 3D reagent was added to the wells. Solutions in the wells were homogenized by pipetting. After an incubation of 30 min in the dark, the relative luciferase units were measured using a GloMax 96 microplate luminometer using the GLOMAX software (version 1.9.2) (Promega). Readouts were normalised to protein concentration per well as measured by Bradford's assay.
7
Dual-Luciferase Reporter Assay Protocol
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Lysis was performed in all cell lines with Passive Lysis Buffer (Promega) and then cells were subjected to a freeze–thaw cycle at −80°C to 37°C and centrifuged at maximum speed for 5 min. The cell lysates were used to determine luciferase and β-gal activities with the Dual-Luciferase Reporter Assay System (Promega) and Beta-Glo Assay System (Promega), respectively, using the Lucy 2 (Anthos Labtec) or GloMax 96 Microplate (Promega) luminometers, according to the manufacturer's standard protocol. When indicated, total protein content was determined with the Pierce 660 nm Assay Kit (Thermo Scientific) using NanoDrop 2000 (Thermo Scientific). The luciferase values are presented as the units of FLuc or RLuc after being normalized to β-gal or, alternatively, per µg of total protein. Each value was derived from at least three independent experiments.
8
Ishikawa Cell Viability Assay
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Ishikawa cells were plated at 2 × 10E3 cells/well in 96-well plates and grown in medium containing 10% FBS for 24 h. After transfection with siRNA, 10 μl of cell count kit-8 (CCK-8, CK04, Dojindo, Japan) was added into each well and cells were incubated for 2 h in a 5% CO2 incubator at 37 °C. The absorbance of each well at 450 nm was read in GloMax™ 96 MICROPLATE (Promega, USA).
9
Cell Proliferation Assay with siRNA
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Ishikawa cells were plated at 2×10E3 cells/well in 96-well plates and grown in medium containing 10% FBS for 24 hrs. After transfection with siRNA , 10 μl of cell count kit-8 (CCK-8, CK04, Dojindo, Japan) was added into each well and cells were incubated for 2 hrs in a 5% CO2 incubator at 37°C. The absorbance of each well at 450 nm was read in GloMax™ 96 MICROPLATE (Promega, USA).
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