For all assays, neutrophils were resuspended in Gibco RPMI 1640 media (Thermo Fisher, Waltham, MA, USA) containing 1% BSA (Sigma-Aldrich, Saint Louis, MO, USA). For the MPO and calcium measurements, the cells were seeded into white flat-bottom 96-well assay plates (Corning Incorporated, New York, NY, USA) at a density of 2 × 105 cells per well. Supernatant sample generation for NE and cfDNA assays was done by seeding cells in a 24-well culture plate (Corning Incorporated, New York, NY, USA), at a concentration of 1 × 106 cells per well. Cells were treated with concentrations of 100 ng/ml, 100 µg/ml and 1 mg/ml anakinra (Swedish Orphan Biovitrum AB, Stockholm, Sweden) at 60, 30, and 10 minutes prior, at the time of and 30 and 60 minutes after stimulation with 100 nM PMA or 5 µg/ml LPS (E.coli O55:B5) as indicated by other studies [25 (link), 27 (link)-29 (link)]. Stimulation was done for 3 hours.
White flat bottom 96 well assay plates
Manufactured by Corning
Sourced in United States
White flat-bottom 96-well assay plates are a standard laboratory equipment used for various experimental assays. These plates provide a uniform surface with 96 individual wells, allowing for multiple experiments or samples to be tested simultaneously. The flat-bottom design ensures consistent liquid volumes and optical clarity, facilitating accurate measurements and data collection.
Automatically generated - may contain errors
Lab products found in correlation
Gibco rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
24 well culture plate, by Corning (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glomax navigator microplate luminometer, by Promega (1 mentions)
Mouse adcc reporter bioassay kit, by Promega (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Acetyltrypsin, by Merck Group (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
Adcc reporter bioassay kit, by Promega (1 mentions)
Adcc effector cells, by Promega (1 mentions)
Fcγriiia v158, by Promega (1 mentions)
Spectramax id3 multi mode microplate reader, by Molecular Devices (1 mentions)
Bio glo luciferase assay reagent, by Promega (1 mentions)
4 protocols using white flat bottom 96 well assay plates
1
Neutrophil Activation Kinetics with Anakinra
2
ADCC Assay for Influenza Antibodies
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To obtain virus-infected cell line, MDCK cells were plated at a density of 2.5 × 104 cells/well in a white flat-bottom 96-well assay plates (Corning) and incubated overnight in a 37°C 5% CO2 incubator. The following day, cells were washed once with PBS and once with OptiMEM (Gibco), and then incubated with X31 (MOI 3) in the presence of 5 μg/ml acetyltrypsin (Sigma) overnight at 34°C.
The X31 HA-expressing EL4 cells were kindly provided by Dr. Garnett Kelsoe (Duke University School of Medicine), and were maintained as previously described [12 (link)].
ADCC activity was determined using a mouse ADCC reporter bioassay kit (Promega) according to manufacturer’s instructions with minor modifications. Briefly, serially diluted mAbs were pre-incubated with the target cell lines for 15 minutes, followed by the addition of effector cells at an effector/target ratio of 3:1. After 6 hours of incubation at 37°C, Bio-Glo Reagent was added for a 30 minutes incubation in the infected condition and for a 15 minutes incubation in HA-expressing condition at room temperature and luminescence was measured by GloMax Navigator Microplate Luminometer (Promega). Irrelevant SARS-CoV-2 specific antibody B38 [71 (link)] was used as a negative control.
The X31 HA-expressing EL4 cells were kindly provided by Dr. Garnett Kelsoe (Duke University School of Medicine), and were maintained as previously described [12 (link)].
ADCC activity was determined using a mouse ADCC reporter bioassay kit (Promega) according to manufacturer’s instructions with minor modifications. Briefly, serially diluted mAbs were pre-incubated with the target cell lines for 15 minutes, followed by the addition of effector cells at an effector/target ratio of 3:1. After 6 hours of incubation at 37°C, Bio-Glo Reagent was added for a 30 minutes incubation in the infected condition and for a 15 minutes incubation in HA-expressing condition at room temperature and luminescence was measured by GloMax Navigator Microplate Luminometer (Promega). Irrelevant SARS-CoV-2 specific antibody B38 [71 (link)] was used as a negative control.
3
Quantifying Antibody-Dependent Cell Cytotoxicity
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The levels of ECA induced by different antibody samples were determined by using a ADCC Reporter Bioassay kit (Promega) according to manufacturing protocol. Briefly, CD20 positive WIL2-S cells were thawed and seeded into white, flat-bottom 96-well assay plates (Corning) at the recommended density. A three-fold serial dilution of each glycoengineered Rituximab was added to the wells. Effector cells were thawed and added into each well at a concentration of 75,000 cells/well, resulting in a target: effector ratio of 1:6. The plate was then incubated for 6 h at 37 °C. Thereafter, Bio-Glo Luciferase Assay Reagent was added into each well and incubated for 10 min before being subjected to luminescence measurement using an Infinite M200 Pro plate reader (Tecan). The levels of ECA were quantified with the luminescence readout.
4
ADCC Assay for Anti-CTLA-4 Antibodies
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ADCC bioassay effector cells in propagation model (Catalog number G7102) with engineered expressions of FcγRIIIA (V158) and an NFAT (nuclear factor of activated T cells)-dependent firefly luciferase reporter gene were purchased from Promega Corporation (Madison, WI, USA). 293T cells with stable expression of OFP-tagged human CTLA-4 were used as the targeted cells. The in vitro ADCC assay was performed following the instructions from the manufacturer of ADCC effector cells (Promega). Briefly, the effector cells (2 × 104 cells/well) were co-cultured with target 293T cells at an effector-to-target ratio of 1:1, and along with indicated concentrations of anti-human CTLA-4 antibodies or control antibody in RPMI medium supplemented with 4% low-IgG serum in white, flat-bottom 96-well assay plates (Corning™). After incubation of plates at 37 °C for 20 h, 50 µL Bio-Glo™ luciferase assay reagent (Promega) was added into each well and the luminescence intensity was measured with a plate reader (SpectraMax iD3 Multi-Mode Microplate Readers, Molecular Devices, LLC, San Jose, CA, USA).
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