Caspase 3/7 activity was measured using the Caspase-Glo® 3/7 assays (Promega Corp., USA) according to manufacturer’s instructions. Protein lysates from ischemic cortex and striatum were subjected to Caspase 3/7 activity measurement with Caspase-Glo assay kit (Promega, Madison USA). 100ul of cell lysates were aliquoted into 1.5ml tubes followed by 100 μl of Caspase-Glo reagent to each well, the content of well was gently mixed with a plate shaker at 300–500 rpm for 30 seconds. The plate was then incubated at room temperature for 3 hours. The luminescence of each sample was measured in a plate-reading luminometer (TECAN, Switzerland) with parameters of 1minute lag time and 0.5 second/well reading time. The experiments were performed in duplicate.
Plate reading luminometer
Manufactured by Tecan
Sourced in Switzerland
The Plate-reading luminometer is a laboratory instrument designed to detect and quantify luminescent signals. It is capable of measuring light emission from samples in multiwell plates, providing quantitative data on various luminescent assays.
Automatically generated - may contain errors
Lab products found in correlation
Caspase glo assay kit, by Promega (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
Caspase 3 7 reagent, by Promega (1 mentions)
Mir 10a 5p mimic, by GenePharma (1 mentions)
Mirna mimic control, by GenePharma (1 mentions)
Prl tk vector, by Promega (1 mentions)
Pmir report luciferase vector, by Thermo Fisher Scientific (1 mentions)
Prl tk renilla, by Promega (1 mentions)
Pmir report luciferase, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter 1000 assay system, by Promega (1 mentions)
E z n a endo free plasmid maxi kit, by Omega Bio-Tek (1 mentions)
Caspase glo 1 inflammasome assay, by Promega (1 mentions)
Gaussia luciferase assay kit, by New England Biolabs (1 mentions)
10 protocols using plate reading luminometer
1
Caspase 3/7 Activity Quantification
2
Identifying and Validating microRNA Targets
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Luciferase reporter transfection and dual luciferase assays were performed following protocols established in our previous reports.26 (link),27 (link) We used the computational target prediction algorithm miRanda (http://cbio.mskcc.org/microRNA2003/miranda.html ) to identify mature gene transcripts that were potentially targeted by JAM-A at their 3′ UTRs. VCAN was then experimentally verified as a JAM-A target gene in hDPCs following the protocols of Wang et al.26 (link),27 (link) Reporter assays used Light Switch 3′ UTR Go Clone Constructs (BioCat GmBH, Heidelberg, Germany) based on the 3′ UTR sequences of JAM-A and VCAN, which were co-transfected with 100 ng of VCAN reporter plasmid and either 10–40 nM miRNA-221–3p mimic or 40 nM control siRNA. After 24 h of incubation, 50 μl of Light Switch reagent (BioCat GmBH, Heidelberg, Germany) was added to each well, and luminescence was measured using a plate-reading luminometer (Tecan, Männedorf, Switzerland). RT–qPCR analysis was performed to quantify the VCAN transcription levels in DP cells at 1 day after incubation with the microRNA-221–3p mimic or inhibitor.
3
Caspase Activity Quantification Assay
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The Caspase-Glo™ 3/7, Caspase-Glo™ 8 or Caspase-Glo™ 9 assay kit was used according to the manufacturer’s instructions to measure the activity of caspase 3/7, caspase-8 or caspase-9, respectively. In brief, 50 μL of cell lysate (cytosolic extracts, 20 μg) was incubated in 50 μL of Caspase-Glo reagent at room temperature for 1 h. After incubation, the luminescence of each sample was measured in a plate-reading luminometer (Tecan, Linz, Austria).
4
Caspase-3/7 Activity Assay
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Cells treated in 96-well plates were mixed with Caspase-3/7 Reagent (Promega, Madison, WI) according to the instructions of the manufacturer and caspase-3/7 activities were assessed using a plate-reading luminometer (Tecan, Männedorf, Switzerland).
5
TGFβRI Kinase Enzyme Assay Protocol
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The TGFβRI Kinase Enzyme System (Promega, Madison, WI, USA) was used in this study. TβRI kinase activity was measured using an in vitro ADP-Glo kinase assay according to the manufacturer’s protocols. TβRI (20 ng/μL), TβRI substrate peptide (200 ng/μL), ATP (25–800 μM), and crizotinib (the indicated concentrations) in kinase assay buffer (Promega) were incubated for 1 h at room temperature. Luminescence was measured using a plate-reading luminometer (Tecan, Männedorf, Switzerland).
6
Cloning and Analysis of TBX5 3'UTR
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TBX5 3′UTR segment was cloned by PCR (Forward primer: GCG GAG CTC GAA ATG AAA CCC AGC ATA; reverse primer: GCG AAG CTT AGC CTC ACA TCT TAC CCT), and then inserted into downstream of the luciferase gene between SacI and HindIII sites within the pMIR‐Report™ luciferase vector (Ambion, Austin, USA). The pRL‐TK vector (Promega, Fitchburg, USA) was used as a control. All plasmid vectors were extracted with the EZNA™ Endo‐free Plasmid Maxi Kit (Omega BioTek, Norcross, USA). The constructed vectors were then sent to the company (GenScript Company, Nanjing, China) for the verification of sequence integrity.
HeLa cells were cultured in a 48‐well plate containing DMEM high‐glucose medium (HyClone, USA) supplemented with 10% FBS (HyClone) 24 hrs before transfection. Then cells (0.5 × 105 cells per well) were transfected with firefly pMIR‐Report™ luciferase (Ambion) and Renilla pRL‐TK (Promega) vectors (90 ng:10 ng per well) and at the same time with 50 nM miRNA control mimic or 50 nM miR‐10a‐5p mimic (GenePharma, China). The Renilla luciferase reporter was used for normalization. After 24 hrs of transfection, the cells were lysed, and luciferase activity was detected using Dual‐Luciferase® Reporter 1000 Assay System (Promega) by a plate‐reading luminometer (Tecan).
HeLa cells were cultured in a 48‐well plate containing DMEM high‐glucose medium (HyClone, USA) supplemented with 10% FBS (HyClone) 24 hrs before transfection. Then cells (0.5 × 105 cells per well) were transfected with firefly pMIR‐Report™ luciferase (Ambion) and Renilla pRL‐TK (Promega) vectors (90 ng:10 ng per well) and at the same time with 50 nM miRNA control mimic or 50 nM miR‐10a‐5p mimic (GenePharma, China). The Renilla luciferase reporter was used for normalization. After 24 hrs of transfection, the cells were lysed, and luciferase activity was detected using Dual‐Luciferase® Reporter 1000 Assay System (Promega) by a plate‐reading luminometer (Tecan).
7
Caspase-3/7 Activity Assay for Apoptosis
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The apoptosis induced by compounds was determined by measuring the activity of caspases-3/7 using Caspase-Glo® 3/ according to the manufacturer’s protocol (Promega, Madison, WI, USA). 10,000 A375 cells were seeded in a 96-well plate at and treated with compounds or DMSO for different time points (24, 48 and 72 h). Afterwards, 100 μL of culture media of each well was transferred to a 96 white multi-well plate and 100 μL of Caspase-Glo® 3/7 reagent was added and incubated for 1 h at room temperature. When the incubation period was completed, the luminescence of each sample representing the enzymatic activity of caspase was measured using a plate-reading luminometer (Tecan, Switzerland). The assay was performed in three independent experiments and each sample was performed in triplicate (mean ± SD, n = 9).
8
Detecting Active Caspase-1 Release
9
Caspase-3/7 Activity Assay for Apoptosis
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The apoptosis induced using compounds was determined by measuring the activity of caspase-3/7 using Caspase-Glo ® 3/ according to the manufacturer's protocol (Promega, Madison, WI, USA). SK-MEL-28 cells were seeded in a 96-well white plate and treated with compounds or DMSO for different time points (12, 24 and 48 h). Afterwards, 100 µL of culture media of each well was transferred to a 96 white multi-well plate, and 100 µL of Caspase-Glo ® 3/7 reagent was added and incubated for one hour at room temperature. When the incubation period was completed, the luminescence of each sample representing the enzymatic activity of caspase was measured using a plate-reading luminometer (Tecan, Männedorf, Switzerland). The assay was performed in three independent experiments, and each sample was performed in triplicate (mean ± SD, n = 9).
10
Wnt/β-Catenin Signaling Pathway Monitoring
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