After an overnight incubation at 37°C, monocytes seeded on 24-well plates (1 × 105 cells/well) were infected with viable CT and CP EBs in SPG buffer (infectivity ratio: 5 EBs/cell) for 60 min in the presence of 10 μM Dichlorofluorescein diacetate (DCFDA) or 5 μM Diaminofluorescein (DAF-2), ROS and RNS probes, respectively. Treatments with 0.8 μM Phorbol or 10 μg/ml Escherichia coli Lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls to induce ROS or RNS production, respectively [27 (link),28 (link)]. Cells were subsequently washed with phosphate buffered saline (PBS) and each well was filled with 300 μl of PBS and glucose (4,5 mg/ml). The increase in cell fluorescence from each well was measured (λexc = 485 nm; λem = 535 nm) with a spectrofluorometer (Wallac Victor multilabel counter, Perkin-Elmer Inc., Boston, MA, USA) at 3, 6 and 24 hours post infection.
Wallac victor multilabel counter
Manufactured by PerkinElmer
Sourced in United States
The Wallac Victor multilabel counter is a versatile instrument designed for high-throughput detection and quantification of various analytes in microplate-based assays. It provides accurate and reliable measurements across a wide range of applications, including fluorescence, luminescence, and absorbance detection. The instrument's core function is to serve as a flexible and efficient platform for researchers and scientists to conduct diverse analytical experiments in their laboratories.
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Lab products found in correlation
4 protocols using wallac victor multilabel counter
1
Chlamydia Infection Induces ROS/RNS
2
Fluorometric Caspase-3 Activity Assay
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The activity of caspase-3-like enzymes (DEVDase activity) was determined fluorometrically as reported previously.59 (link) Briefly, cells were scraped and centrifuged at 300 × g at 4 °C for 5 min. Pellets were washed in ice-cold PBS, re-suspended in PBS and then flash-frozen in liquid nitrogen. Fifty micromoles of DEVDase-substrate (DEVD-MCA) in reaction buffer (100 mM HEPES pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 5 mM DTT, 0.01% NP-40) was added to the lysates. The release of free AMC was monitored at 37 °C at 60-s intervals over a 30-min period using a Wallac Victor multilabel counter (Perkin Elmer, Ballymount, Ireland) (excitation 355 nm, emission 460 nm). Fluorescent units were converted to nanomoles of AMC released per minute per mg of enzyme using a standard curve generated with free AMC and subsequently related to protein concentration.
3
Cytotoxicity Assessment of Peptides
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Cytotoxicity of peptides was estimated using a MTT assay. All the chemicals used for this assay have been purchased by sigma Aldrich and used without further purification. T67, HeLa and HDF cells were seeded in 24-well plates at 1 × 105 cells/well. Experiments were performed after 24 h of incubation at 37 °C in 5% CO2. After this time cells were washed and treated for 24 h with different concentrations of peptides; the cells are then incubated for 60 mins with 300 μM of MTT in culture medium. The absorbance of each well was measured at 450 nm with a spectrofluorometer (Wallac Victor multilabel counter; Perkin-Elmer Inc., Boston, MA, USA). Data are reported as the mean ± standard deviation of at least three independent experiments50 .
4
PD-1/PD-L1 Binding Inhibition Assay
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CHO cells expressing human or cynomolgus PD-1 were incubated for 1 h at 6 × 104 cells/well with serially diluted ezabenlimab or controls, and 0.22 µg/mL biotin-labeled hPD-L1-Fc chimera or 0.33 µg/mL biotin-labeled hPD-L2-Fc chimera (R&D Systems, biotinylated at Boehringer Ingelheim). Cells were washed and DELFIA Eu-labeled streptavidin was added for a 1-h incubation period. Cells were washed and DELFIA Enhancement solution was added prior to 30 min incubation. Fluorescence was measured using Wallac VICTOR Multilabel Counter (Perkin Elmer, 1420). Data were normalized to percent inhibition where 0% inhibition equaled the fluorescence value of cells incubated with the ligand without antibodies, and 100% inhibition equaled the signal obtained with cells without labeled ligands or antibodies. Results were excluded if the 95% confidence interval factor (upper/lower) was >5.
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