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.
Caspase glo 1 assay
Manufactured by Promega
Sourced in United States
The Caspase-Glo 1 Assay is a bioluminescent, homogeneous assay designed to measure caspase-1 activity in cell-based and cell-free systems. The assay uses a luminogenic caspase-1 substrate which, upon cleavage by the enzyme, generates a luminescent signal proportional to the amount of caspase-1 activity present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax i3 system, by Molecular Devices (2 mentions)
Luciferin wehd substrate, by Promega (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (2 mentions)
Anti lc3, by Cell Signaling Technology (2 mentions)
Anti mda5, by Cell Signaling Technology (2 mentions)
Anti β actin hrp, by Abcam (2 mentions)
Anti caspase 1, by Thermo Fisher Scientific (2 mentions)
Fam yvad fmk, by ImmunoChemistry Technologies (1 mentions)
Accuri c6, by BD (1 mentions)
Spectramax i3x multi mode microplate, by Molecular Devices (1 mentions)
Anti neun antibody ab177487, by Abcam (1 mentions)
Anti β actin 3700, by Cell Signaling Technology (1 mentions)
Anti histone h3 antibody 4499, by Cell Signaling Technology (1 mentions)
Quantinova sybr green pcr kit, by Qiagen (1 mentions)
7 protocols using caspase glo 1 assay
1
SARS-CoV-2 Infection of Human Monocytes: Caspase-1 and LDH Assay
2
Evaluating Caspase-1 Activation in COVID-19
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To evaluate caspase-1 activation, 5 × 105 PBMCs from COVID-19 patients or healthy donors were centrifuged (400 ×g, 10 min), and cells were labeled for 30 min with the FLICA carboxyfluorescein reagent (FAM–YVAD–FMK; Immunochemistry Technologies), as recommended by the manufacturer. The cells were then washed two times with PBS 1× and fixed with fixative reagent provided by the manufacturer. Acquisition was performed in fixed cells in a flow cytometer (BD Accuri C6; BD Biosciences) and then analyzed using FlowJo (Tree Star) software. To evaluate caspase-1 activity in supernatants, 2 × 105 PBMCs from COVID-19 patients were plated in 96-well plates and incubated overnight. To measure caspase-1 activity, the supernatants were collected, and the Caspase-Glo 1 Assay (Promega) was performed following the manufacturer’s instructions.
3
Caspase-1 Activity Assay in Macrophages
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The Caspase-Glo 1 assay (Promega) was used to measure the activity of caspase-1 in J774.1 macrophages either infected with M. tuberculosis at an MOI of 5 or treated with nigericin, an NLRP3 inflammasome inducer, at a final concentration of 20 μM. After 4–6 hours of infection or nigericin treatment, unprocessed cell samples were mixed at a 1:1 volume ratio with assay reagent in 96-well opaque plates, and the activity of caspase-1 in each sample was measured according to the manufacturer’s protocol. In brief, after 90 minutes of incubation at room temperature, luminescence produced by caspase cleavage of the Z-WEHD substrate was read on a standard plate reader. A caspase-1–specific inhibitor (Ac-YVAD-CHO) was added to parallel samples to confirm specific activity, and wells without cells were run to control for background signal. The values from both the Ac-YVAD-CHO and blank control were subtracted from the Z-WEHD substrate sample value, representing caspase-1 activity.
Immunoblots of cell lysates were probed with the following antibodies: anti-MDA5 (Cell Signaling Technology 3743), anti-LC3 (Cell Signaling Technology 12741), anti–caspase-1 (eBioscience 5B10), or anti–β-actin–HRP (Abcam 49900).
Immunoblots of cell lysates were probed with the following antibodies: anti-MDA5 (Cell Signaling Technology 3743), anti-LC3 (Cell Signaling Technology 12741), anti–caspase-1 (eBioscience 5B10), or anti–β-actin–HRP (Abcam 49900).
4
Caspase-1 Activity Assay in Macrophages
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The Caspase-Glo 1 assay (Promega) was used to measure the activity of caspase-1 in J774.1 macrophages either infected with M. tuberculosis at an MOI of 5 or treated with nigericin, an NLRP3 inflammasome inducer, at a final concentration of 20 μM. After 4–6 hours of infection or nigericin treatment, unprocessed cell samples were mixed at a 1:1 volume ratio with assay reagent in 96-well opaque plates, and the activity of caspase-1 in each sample was measured according to the manufacturer’s protocol. In brief, after 90 minutes of incubation at room temperature, luminescence produced by caspase cleavage of the Z-WEHD substrate was read on a standard plate reader. A caspase-1–specific inhibitor (Ac-YVAD-CHO) was added to parallel samples to confirm specific activity, and wells without cells were run to control for background signal. The values from both the Ac-YVAD-CHO and blank control were subtracted from the Z-WEHD substrate sample value, representing caspase-1 activity.
Immunoblots of cell lysates were probed with the following antibodies: anti-MDA5 (Cell Signaling Technology 3743), anti-LC3 (Cell Signaling Technology 12741), anti–caspase-1 (eBioscience 5B10), or anti–β-actin–HRP (Abcam 49900).
Immunoblots of cell lysates were probed with the following antibodies: anti-MDA5 (Cell Signaling Technology 3743), anti-LC3 (Cell Signaling Technology 12741), anti–caspase-1 (eBioscience 5B10), or anti–β-actin–HRP (Abcam 49900).
5
Caspase-1 Activity Assay Protocol
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To measure caspase-1 activity, the supernatants were collected and incubated with the Luciferin WEHD substrate provided by the Caspase-Glo 1 assay (Promega, WI, USA). After 1 hour of incubation at room temperature, luminescence was measured using SpectraMax i3x Multi-Mode Microplate (Molecular Devices).
6
SARS-CoV-2 Infection and Caspase-1 Activity in hACE2-Expressing Macrophages
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THP-1 derived macrophages stably overexpressing hACE2 (4 × 105 cells) were plated on 24-well plates in RPMI-1640 medium supplemented with 10% FBS and incubated overnight. Cells were then infected with SARS-CoV-2 at an MOI of 1 in RPMI-1640 culture medium without Phenol Red for 1 h to allow virus adsorption. After virus inoculation, fresh RPMI-1640 medium supplemented with 2% FBS with or without 15 μM MCC950 were added for 24 h. To determine caspase-1 activity, culture supernatants were collected and incubated with the luciferin WEHD-substrate in the Caspase-Glo 1 Assay (Promega). After an incubation for 1 h at room temperature, luminescence was detected 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.
7
Molecular Mechanisms of Inflammasome Activation
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Bilirubin (B4126), ethidium bromide (EtBr) (E1385), and 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (M5655) were purchased from Sigma-Aldrich, Co. (St. Louis, MO). Ethidium homodimer-2 (EthD2) (E3599) was purchased from Life Technologies (Eugene, OR). VX-765 (S2228) was purchased from Selleck Chemicals (Houston, TX). The Caspase-Glo 1 assay was purchased from Promega (Madison, WI). Anti-Gasdermin D (anti-GSDMD) antibody (93709), anti-nuclear factor-kappa B (NF-κB) p65 antibody (8242), anti-Histone H3 antibody (4499), and anti-β-actin (3700) were purchased from Cell Signaling Technology (Danvers, MA). Anti-NeuN antibody (ab177487) was purchased from Abcam (Cambridge, UK). The nonradioactive infrared NF-κB electrophoretic mobility shift assay (EMSA) Kit was purchased from Viagen Biotech (Changzhou, China). The enzyme-linked immunosorbent assay (ELISA) kits for IL-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α were purchased from Raybiotech (Norcross, GA). Quanti-Nova SYBR Green PCR Kit (208054) was purchased from Qiagen (Hilden, Germany).
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