Caspase-3 activity was evaluated using Caspase Fluorescent (AFC) Substrate/ Inhibitor QuantiPak (ENZO Life Sciences), following the manufacturer protocol. Briefly, cell lysates (total protein, 100 μg) were added to reaction mixtures (final volume, 100 μl) containing fluorigenic substrate peptides that are specific for caspase 3 (DEVD-AFC). The reaction was performed at 37 °C for 1 h. Fluorescence was measured with a fluorescence microplate reader (Microplate Imaging System, Bio-Rad), at 530 nm.
Microplate imaging system
Manufactured by Bio-Rad
Sourced in United States
The Microplate Imaging System is a versatile laboratory instrument designed for high-throughput imaging and analysis of multi-well microplates. It provides accurate and consistent results by capturing detailed images of samples within microplate wells.
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Lab products found in correlation
6 protocols using microplate imaging system
1
Caspase-3 Activity Quantification
2
Determining Cytotoxic Efficacy of 5FU and PTX
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KB-3-1, KB-8-5, H460, H460/Tax-R or NIH/3T3 fibroblast cells were seeded into 96-well plates at a density of 1× 104 cells per well and allowed to adhere overnight. To determine the IC50 of 5FU for different cell lines, NIH/3T3, KB-8-5 and KB-3-1 cell were subject to 5FU treatment (from 0.1 μM to 50 μM). To determine the killing efficiency of combination therapy, various concentrations of PTX (from 0.1 nM to 100 nM) were added to the wells in the absence or presence of 5 μM 5FU for 48 h. Following incubation, 20 μl of MTT reagent (Sigma-Aldrich, St. Louis, MO) (5mg/ml in PBS) was added to the culture medium and the cells were incubated for an additional 4 h at 37°C. Then the culture media were carefully removed and 200 μl of DMSO were added to the wells to dissolve the formazan. Plates were read at 570 nm on a microplate reader using a Bio-Rad microplate imaging system (Hercules, CA) and results were expressed as cell viability (%) calculated as (OD of treated group / OD of control group) × 100.
3
Caspase-3 Activity Quantification
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Caspase-3 activity was evaluated using Caspase Fluorescent (AFC) Substrate/Inhibitor QuantiPak (ENZO Life Sciences) following the manufacturer's protocol, and Microplate Imaging System (Bio-Rad) performed the measurement of enzymatic activity at 530nm. The means and standard deviations were calculated from two independent experiments.
4
Cell Proliferation Assay with Ruxolitinib
5
Metabolic Biomarker Analysis Protocol
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Plasma glucose concentration was measured colorimetrically (glucose oxidase-phenol and 4
aminophenazone (GOD-PAP) method; Roche Diagnostics) and analysed using a micro plate
imaging system (Bio-Rad Laboratories, Inc.). Insulin concentration was measured using
ELISA (DRG) according to the manufacturer’s protocol. Homoeostasis model assessment of
insulin resistance as calculated from fasting plasma glucose and insulin concentrations
(glucose (mmol/l)×insulin (pmol/l)/22·5) as an indirect measure of insulin sensitivity.
Total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol and TAG
concentrations were determined colorimetrically after enzymatic conversion using a Roche
Hitachi 717 analyzer (Reinier de Graaf Laboratory).
aminophenazone (GOD-PAP) method; Roche Diagnostics) and analysed using a micro plate
imaging system (Bio-Rad Laboratories, Inc.). Insulin concentration was measured using
ELISA (DRG) according to the manufacturer’s protocol. Homoeostasis model assessment of
insulin resistance as calculated from fasting plasma glucose and insulin concentrations
(glucose (mmol/l)×insulin (pmol/l)/22·5) as an indirect measure of insulin sensitivity.
Total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol and TAG
concentrations were determined colorimetrically after enzymatic conversion using a Roche
Hitachi 717 analyzer (Reinier de Graaf Laboratory).
6
Cell Viability Assay Using MTT
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KB-3-1 and KB-8-5
cells were seeded into 96-well plates at a density of 1 × 104 cells per well and allowed to adhere overnight. Various concentrations
of drug or formulation were added to the plates for 48 h. Following
incubation, 20 μL of MTT reagent (5 mg/mL in PBS) was added
to the culture medium and the cells were incubated for an additional
4 h at 37 °C. The culture medium was then carefully removed,
and 200 μL of DMSO was added to the wells to dissolve the formazan.
UV absorbance was measured at 570 nm using a Bio-Rad microplate imaging
system (Hercules, CA), and results were expressed as % cell viability
(OD of treated group/OD of control group × 100).
cells were seeded into 96-well plates at a density of 1 × 104 cells per well and allowed to adhere overnight. Various concentrations
of drug or formulation were added to the plates for 48 h. Following
incubation, 20 μL of MTT reagent (5 mg/mL in PBS) was added
to the culture medium and the cells were incubated for an additional
4 h at 37 °C. The culture medium was then carefully removed,
and 200 μL of DMSO was added to the wells to dissolve the formazan.
UV absorbance was measured at 570 nm using a Bio-Rad microplate imaging
system (Hercules, CA), and results were expressed as % cell viability
(OD of treated group/OD of control group × 100).
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