Supernatant from 5 × 105 BMDMs was collected, and the activity of released LDH was measured using colorimetric assays with CytoTox 96® (Promega) according to the manufacturer’s instructions. The percentage of LDH release was calculated as the ratio of the OD 490-nm sample/maximum OD. A maximum was obtained from BMDMs lysed with Triton X100. The OD values were read on SpectraMax i3 system (Molecular Devices).
Spectramax i3 system
Manufactured by Molecular Devices
Sourced in United States, Brazil
The SpectraMax i3 system is a multimode microplate reader that offers high-performance detection capabilities. It is designed to provide accurate and reliable measurements for a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Caspase glo 1 assay, by Promega (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Cytotox 96, by Promega (1 mentions)
Luciferin wehd substrate, by Promega (1 mentions)
Lps escherichia coli 055 b5, by Merck Group (1 mentions)
Opteia kit, by BD (1 mentions)
96 well flat clear bottom black polystyrene tc treated microplates, by Corning (1 mentions)
Bio glo reagent, by Promega (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
3 3 5 5 tetramethylbenzidine substrate solution, by Solarbio (1 mentions)
14 protocols using spectramax i3 system
1
Quantifying BMDM Cytotoxicity by LDH Release
2
SARS-CoV-2 Infection of Human Monocytes: Caspase-1 and LDH Assay
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For caspase-1 and LDH determination, 2 × 105 human CD14+ monocytes were plated on 48-well plates in RPMI 10% FBS and incubated overnight. The following day, cells were infected with SARS-CoV-2 using MOI 5 in RPMI without Phenol Red (3.5 g/liter Hepes, 2 g/liter NaHCO3, 10.4 g/liter RPMI without Phenol Red, and 1% glutamine, pH 7.2). After 1 h of virus adsorption, fresh media (RPMI 2% FBS without Phenol Red) with or without MCC950 at 10 µM were added, and cells were incubated for 24 h. To measure caspase-1 activity, the supernatants were collected and incubated with the Luciferin WEHD-substrate provided by the Caspase-Glo 1 Assay (Promega). After 1 h of incubation at room temperature, luminescence was measured using the SpectraMax i3 system (Molecular Devices). LDH release was measured in the supernatants using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) following the manufacturer’s instructions.
3
Quantifying Cytokine Levels by ELISA
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For the quantification of cytokines by ELISA (Enzyme-Linked ImmunoSorbent Assay), BMDMs were plated in 24-well plates (5 × 105 cells/well) and pretreated with 100 ng/ml LPS (Escherichia coli 055: B5 LPS, Sigma) or Pam3Cys for 4 h and then infected with L. amazonensis (MOI 10). The cytokine IL-1β present in the supernatant was measured by ELISA using the BD OptEIA kits (BD Biosciences), according to instructions provided by the manufacturer. The OD values were read on the SpectraMax i3 system (Molecular Devices).
4
Quantifying Inflammatory Cytokines by ELISA
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The levels of IL-1β and IL-18 in the supernatants from cultured cells were measured by commercial ELISA assay (R&D Systems) following the manufacturer's instructions. ELISA values were determined using the SpectraMax i3 system (Molecular Devices).
5
Pseudomonas aeruginosa Growth Kinetics
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Overnight cultures of P. aeruginosa were diluted 1:1,000 in 150 μL volumes of sterile LB media in 96-well microtiter plate wells. Plates were incubated at 37°C and optical density (OD) at 600 nm was measured every 15 min using a SpectraMax i3 system (Molecular Devices, CA, USA). Plates were shaken prior to each measurement. Assays were performed in biological duplicate and the mean was used to generate growth curves. To provide a measure of growth rate, a 3-h period within the exponential phase of growth (between 5.5 h and 8.5 h) was used to infer the change in OD per hour using the following equation: ΔOD600/hr = OD600 at 8.5 h − OD600 at 5.5 h/3.
6
Measuring Intracellular Potassium Dynamics
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Intracellular potassium concentrations were determined by fluorescence emission from the Assante Potassium Green-2 probe (APG-2 AM, TEFlabs) as previously described66 (link). BMDMs from C57BL/6 and Gsdmd–/– mice were plated in 96-well plates (Corning 96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates) and infected by L. amazonensis for 2 or 24 h. After infection, the supernatant was removed and cultures were added in RMPI 1640 medium without phenol red and without SFB, containing 5 μM APG-2 AM for 30 min. After incubation with the probe, the wells were washed 3 times with PBS at room temperature and added to the end of the RPMI 1640 washes without phenol red and without SFB. Fluorescence was read on the SpectraMax i3 system (Molecular device) fluorometer. The parameters of the analysis of the Relative Fluorescence Units (RFU), were standardized to read the emission in the wavelength determined by the manufacturer.
7
ELISA for Antibody Binding Affinity
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Human 4-1BB, GITR, OX40 or CD40 (ACRO Biosystems) were coated onto a 96-well plate overnight at 4°C. The plate was washed and blocked for 1 hour in 5% bovine serum albumin/phosphate buffered saline (BSA/PBS) at room temperature. Serially diluted antibodies were added to the plate in duplicate and incubated for 1 hour at room temperature. After washing, Goat (ab’)2 antihuman IgG Fc (HRP) (Abcam) was added and incubated at room temperature for 1 hour. Plates were washed and signal developed using 3,3’,5,5’-tetra-methylbenzidine substrate solution (Solarbio) according to manufacturer’s instructions. Absorbance at 450 nm was read on a Molecular Devices SpectraMax i3 system with SoftMax Pro software.
8
CRF Quantification in Serum Samples
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Experiments were performed at the Biofluid Biomarkers Core of the Cohen Veteran Center in Dr. Fossati's lab at NYULMC. Operators performing assays were blind to study group. Barcode readers were used to allow for sample blinding. CRF was measured in serum samples by an enzyme immunoassay (EIA) (Phoenix Pharmaceuticals Inc.). Absorbance was detected using a Spectra Max i3 system (Molecular Devices). Limit of detection (LOD) for the assay is 0.33 ng/ml, Intra‐assay variation is below 10%, and Inter‐assay variation, below <15%. The assay is specific for human CRF, with no cross‐reactivity for human Prepro‐CRF (125‐151), PACAP‐38, LH‐RH, ACTH or [Arg8]‐Vasopressin. All serum samples were well above the LOD and within the levels expected from the assay specifications. All assays were run in duplicate. Previously obtained serum aliquots from healthy donors were included in all experiments and used for batch normalization. Samples showing coefficients of variation higher than 20% were excluded and measured again.
9
PD-L1 Receptor Activation Assay
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PD-L1-negative or PD-L1-positive CHO cells were co-cultured with Jurkat-hu4-1BB-NF-κB-Luc cells in the presence of 1 µg/mL OKT-3 and serially diluted antibodies for 16 hours at 37°C. Bio-Glo Reagent (Promega), prepared according to manufacturer’s instructions, was added and fluorescence measured using a SpectraMax i3 system with SoftMax Pro software (Molecular Devices).
10
Plasma Lipid and Glucose Analysis
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Plasma concentrations of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), TG, and glucose were determined using commercial kits (Gold Análise Diagnostica Ltda, MG, Brazil) and the SpectraMax i3 system (Molecular Devices, Sunnyvale, CA, USA).
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