In all, ~105 tumor cells were co-incubated with PBMCs from HLA-A2-negative healthy individuals in 96-well plates (Costar®, Corning Inc., USA) and incubated for 20 h using standard cell culture conditions (37 °C, 5% CO2) in the presence of constructs as indicated. Intracellular luciferase activity was monitored to determine killing of the firefly luciferase-expressing THP-1 tumor cells in the presence of HLA-A2 negative PBMCs and constructs. d -Luciferin (Biosynth Inc., USA) was added to a final concentration of 0.5 mM and incubated at 37 °C for 20–30 min. Subsequently, light emission was quantified with an infinite M200 pro ELISA reader (Tecan Ltd, Switzerland). Ten percent DMSO was used to assess maximal killing, which was set at 100%.
Infinite m200 pro elisa reader
Manufactured by Tecan
Sourced in Switzerland, Germany
The Infinite M200 pro ELISA reader is a laboratory instrument designed for performing enzyme-linked immunosorbent assays (ELISA). It is capable of measuring absorbance in microplates, with a wavelength range of 230 to 1000 nm. The Infinite M200 pro provides precise and reliable data for a variety of ELISA-based applications.
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4 protocols using infinite m200 pro elisa reader
1
Quantifying Tumor Cell Killing by PBMCs
2
Mitochondrial Energy Metabolism Kinetics
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To evaluate ATP production kinetics, isolated liver mitochondria were diluted in 100 µL ice cold assay buffer (125 mM KCl, 2 mM K2HPO4, 20 mM HEPES, 1 mM MgCl2, 100 µM EGTA, 0.025% BSA, pH 7.0) supplemented with 2.5 mM malate, 2.5 mM glutamate, 2.5 mM succinate, and 3 mM ADP. Control reactions were additionally supplemented with 2.5 µM oligomycin to inhibit ATP synthase activity and 2.5 µM FCCP to uncouple respiration and ATP synthase activity. Samples were incubated for 30 min at 37 °C. Afterwards, to detect the generated ATP content, the reaction mix was supplemented with 100 µL CellTiter-Glo Luminescent Cell Viability Assay reaction buffer (Promega, Mannheim, Germany). Chemiluminescence was detected using the Infinite M200 PRO ELISA reader (Tecan Deutschland GmbH, Crailsheim, Germany).
Mitochondrial superoxide was determined using MitoSOX Red (Invitrogen, Thermo Fisher Scientific, Schwerte, Germany). Reactions were supplemented with 200 nM MitoSOX Red. Reactions were incubated at 37 °C and MitoSOX Red fluorescence was evaluated at the following wavelength settings: excitation: 510 nm and emission: 580 nm on the Infinite M200 PRO ELISA reader (Tecan Deutschland GmbH, Crailsheim, Germany).
Hydrogen peroxide levels in liver mitochondria were measured using Amplex Red HRP kit (Invitrogen, Thermo Fisher Scientific, Schwerte, Germany), according to the manufacturer’s protocol.
Mitochondrial superoxide was determined using MitoSOX Red (Invitrogen, Thermo Fisher Scientific, Schwerte, Germany). Reactions were supplemented with 200 nM MitoSOX Red. Reactions were incubated at 37 °C and MitoSOX Red fluorescence was evaluated at the following wavelength settings: excitation: 510 nm and emission: 580 nm on the Infinite M200 PRO ELISA reader (Tecan Deutschland GmbH, Crailsheim, Germany).
Hydrogen peroxide levels in liver mitochondria were measured using Amplex Red HRP kit (Invitrogen, Thermo Fisher Scientific, Schwerte, Germany), according to the manufacturer’s protocol.
3
Nanoluciferase Protein Interaction Assay
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To perform luciferase-based protein interaction analysis, a Nano-BiT PPI Starter Systems Kit was used (Promega). Vectors including recombinant luminescence complementation tags LgBiT and SmBiT were fused to survivin and DNA-PKcs PI3K domain sequences and transfected into HEK293T cells using the Effectene transfection protocol (Qiagen). At 1 hour after a 4 Gy irradiation, Nano-Glo live-cell reagent was added and luminescence was measured with an Infinite M200 Pro Elisa Reader (TECAN).
4
SARS-CoV-2 Neutralization Antibody Detection
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The GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (Genscript Biotech, Leiden, The Netherlands) was performed according to the manufacturer's instructions. Briefly, serum samples as well as negative and positive controls were diluted 1:10 in sample dilution buffer, mixed 1:1 with HRP-RBD working solution and incubated at 37 °C for 30 min. Subsequently, 100 µL of sample and controls were added into the wells of the 96-well plate and coated with the ACE2 receptor. The plate was incubated at 37 °C for 15 min and washed 4 × with 300 µL of washing buffer. Next, 100 µL of substrate solution were added and the plate was incubated in the dark for 15 min at RT. Finally, 50 µL of stop solution per well were added and the absorption at 450 nm was measured by using a Tecan infinite M200 Pro ELISA-Reader. The percentage of signal inhibition in relation to the negative control was calculated as follows: Inhibition [%] = (1 – (Sample OD450/Average Negative Control OD450)) × 100. All samples and controls were analyzed in duplicate. The analysis was repeated if the coefficient of variation between both replicates was above 10%.
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