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Caspase 3 colorimetric assay kit

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The Caspase-3 Colorimetric Assay kit is a laboratory tool designed to detect and measure the activity of the enzyme caspase-3. Caspase-3 is a key executioner in the apoptosis (programmed cell death) pathway. The kit utilizes a colorimetric substrate that, when cleaved by active caspase-3, produces a colored product that can be quantified using a spectrophotometer.

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Lab products found in correlation

Protease inhibitor cocktail, by Merck Group (2 mentions) Donepezil hydrochloride, by Merck Group (1 mentions) Trichloracetic acid, by Merck Group (1 mentions) Dithiobisnitrobenzoic acid, by Merck Group (1 mentions) Acetylthiocholine iodide, by Merck Group (1 mentions) Nitroblue tetrazolium, by Merck Group (1 mentions) Colchicine, by Merck Group (1 mentions) Piperine, by Thermo Fisher Scientific (1 mentions) Folin phenol reagent, by Merck Group (1 mentions) Thiobarbituric acid, by Merck Group (1 mentions) Urea assay kit, by Diamond Diagnostics (1 mentions) Poloxamer 407, by BASF (1 mentions) Cremophor rh40, by BASF (1 mentions) N acetylcysteine nac, by Merck Group (1 mentions) Jc 1 probe, by Merck Group (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Cell death detection elisa kit, by Roche (1 mentions) Tsp1 knockout mice, by Jackson ImmunoResearch (1 mentions) Palmitate, by Merck Group (1 mentions)

31 protocols using caspase 3 colorimetric assay kit

1

Caspase-3 Activity Quantification in Diabetic Retina

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The caspase-3 colorimetric assay kit (R&D Systems, Minneapolis, MN, USA) measured the increased enzymatic activity of the caspase-3 class of proteases in retinal tissue as per manufacturer’s instructions. Briefly, the enzymatic reaction for caspase activity was carried out by the addition of 250 μg protein/50 μl of rat retinal homogenate in a 96 well microplate. The cleavage of caspase-3 colorimetric substrate (DEVD-pNA) by the caspase, releases the chromophore pNA, which was quantitated spectrophotometrically at a wavelength of 405 nm using microplate reader (Auto Bio Labtech Instruments, Co, Ltd, China). The results are expressed as fold increase in caspase activity as represented by an increase in optical density in diabetic retina over control retina.
Ola M.S., Ahmed M.M., Shams S, & Al-Rejaie S.S. (2016). Neuroprotective effects of quercetin in diabetic rat retina. Saudi Journal of Biological Sciences, 24(6), 1186-1194.
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2

Caspase-3 Assay in Bladder Cancer

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Briefly, bladder cancer cells T24 and 5637 were plated at 5 ×105 cells/well in a 6-well plate for 24 h, then transfected with corresponding pcDNA-CASC2 or empty vector, respectively. At 48 h after transfection, cleaved caspase-3 activity were measured with the Caspase-3 Colorimetric Assay kit (R&D Systems, Minneapolis, USA) according to the manufacturer's recommendations. The assays were performed in triplicates.
Pei Z., Du X., Song Y., Fan L., Li F., Gao Y., Wu R., Chen Y., Li W., Zhou H., Yang Y, & Zeng J. (2017). Down-regulation of lncRNA CASC2 promotes cell proliferation and metastasis of bladder cancer by activation of the Wnt/β-catenin signaling pathway. Oncotarget, 8(11), 18145-18153.
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3

Formulation and Evaluation of Piperine-Loaded Colloidal Systems

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Piperine (molecular weight =285.34 Da, purity 98%) was purchased from Alpha Aesar (Ward Hill, MA, USA); Peceol® (GMO, hydrophilic-lipophilic balance (HLB) =3) was a kind gift from Gattefosse Co. (Lyon, France); Cremophor RH 40® (Polyoxy 40 hydrogenated castor oil, HLB =14–16) and Poloxamer 407 were obtained from BASF Co. (Ludwigshafen, Germany); Tween 80 (HLB =15) was obtained from El-Nasr Pharmaceutical Co. (Abu Zaabal, Egypt). Colchicine, donepezil hydrochloride, thiobarbituric acid (TBA), trichloracetic acid, nitroblue tetrazolium (NBT), acetylthiocholine iodide, dithiobisnitrobenzoic acid, Folin phenol reagent, protease inhibitor cocktail, and reagents of in vivo study were obtained from Sigma-Aldrich (St Louis, MO, USA). Caspase-3 colorimetric assay kit and tumor necrosis factor-α (TNF-α) Immunoassay kit were purchased from R&D Systems Inc., Wiesbaden-Nordenstadt, Germany. alanine amin otransferase (ALT) and aspartate aminotransferase (AST) assay kits were purchased from Spectrum (Hannover, Germany). Urea assay kit was purchased from Diamond Diagnostics (Hannover, Germany). Creatinine assay kit was purchased from Spectrum. All other chemicals and reagents used were of analytical grade.
Elnaggar Y.S., Etman S.M., Abdelmonsif D.A, & Abdallah O.Y. (2015). Novel piperine-loaded Tween-integrated monoolein cubosomes as brain-targeted oral nanomedicine in Alzheimer’s disease: pharmaceutical, biological, and toxicological studies. International Journal of Nanomedicine, 10, 5459-5473.
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4

Ovarian Caspase-3 Activity Quantification

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Immunohistochemical detection of Cyto c was carried out. In addition, ovarian caspase-3 activity was assessed using caspase-3 colorimetric assay Kit (R & D systems, Inc.) according to the manufacturer's instructions. Briefly, this assay quantifies caspase-3 activation by measuring the cleavage of caspase-3 substrate; DEVD-pNA releasing the chromophore pNA, which can be measured using a microplate reader at a wavelength of 405 nm. The level of caspases-3 enzymatic activity was directly proportional to the color reaction and was expressed as nmol/mg protein. Protein was determined according to Lowry method [31 (link)].
Mahran Y.F., El-Demerdash E., Nada A.S., El-Naga R.N., Ali A.A, & Abdel-Naim A.B. (2015). Growth Hormone Ameliorates the Radiotherapy-Induced Ovarian Follicular Loss in Rats: Impact on Oxidative Stress, Apoptosis and IGF-1/IGF-1R Axis. PLoS ONE, 10(10), e0140055.
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5

Measuring Caspase-3 Activity in DPSCs

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After culturing the cells as described above under 4.4.1, Caspase-3 activity of the GFP-DPSCs and Bcl-2-DPSCs was measured using Caspase-3 Colorimetric Assay kit (R&D Systems, Inc., Minneapolis, MN, USA). Briefly, the Lysis Buffer was added to collect the whole-cell lysate. Then, the cell lysate was incubated on ice for 10 min, centrifuged and the supernate was transferred to a new tube. After adding 50 μL of cell lysate, 50 μL of 2× Reaction Buffer, and 5 μL of Caspase-3 colorimetric substrate to each well, the microplate was incubated for 2 h at 37 °C. Using a microplate reader, readings were taken at wavelength of 405 nm. No cell lysate and no substrate groups were used as controls of the assay.
Dissanayaka W.L., Han Y., Zhang L., Zou T, & Zhang C. (2020). Bcl-2 Overexpression and Hypoxia Synergistically Enhance Angiogenic Properties of Dental Pulp Stem Cells. International Journal of Molecular Sciences, 21(17), 6159.
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6

Caspase-3 Activity Quantification

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Caspase-3 activity was estimated using caspase-3 colorimetric assay kit (R&D Systems. Inc, USA). Results were expressed as nmol p-nitroanilide (pNA)/h/mg protein.
Ahmed L.A., Shehata N.I., Abdelkader N.F, & Khattab M.M. (2014). Tempol, a Superoxide Dismutase Mimetic Agent, Ameliorates Cisplatin-Induced Nephrotoxicity through Alleviation of Mitochondrial Dysfunction in Mice. PLoS ONE, 9(10), e108889.
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7

Dihydroartemisinin-Induced Apoptosis Mechanisms

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DHA (Cat: A2679) was purchased from LKT-Laboratory. Inc., Minnesota, USA. MTT (Cat: M5655), dimethyl sulfoxide (DMSO) (Cat: PHR1309), JC-1 probe (Cat: T4069), N-acetylcysteine (NAC) (Cat: 1009005) were from Sigma-Aldrich Munich, Germany. Annexin V-FITC/PI apoptosis detection kit (Cat: 4830-01-K), Caspase-3 Colorimetric Assay kit (Cat: BF3100) were purchased from R & D System. Marker Gene™ Live Cell Fluorescent ROS Detection Kit (Cat: M1049), Antibodies against Bcl2 (sc-7382), Bax (sc-13156), cytochrome c (Cat: Sc-13561), and also horseradish peroxidase secondary antibodies (sc-358923) were purchased from Santa Cruz Biotechnology Co.
Poupel F., Aghaei M., Movahedian A., Jafari S.M, & Shahrestanaki M.K. (2017). Dihydroartemisinin Induces Apoptosis in Human Bladder Cancer Cell Lines Through Reactive Oxygen Species, Mitochondrial Membrane Potential, and Cytochrome C Pathway. International Journal of Preventive Medicine, 8, 78.
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8

Apoptosis Measurement Protocols

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Apoptosis was measured using a Cell Death Detection ELISA kit (Roche Diagnostics) for measuring DNA fragmentation and the Caspase-3 Colorimetric Assay kit (R&D Systems) for determining enzymatic activity of the caspase-3 in apoptotic cells, as described previously [16 (link),17 (link)].
Ko S.H., Choi J.H, & Kim J.M. (2023). Bacteroides fragilis Enterotoxin Induces Autophagy through an AMPK and FoxO3-Pathway, Leading to the Inhibition of Apoptosis in Intestinal Epithelial Cells. Toxins, 15(9), 544.
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9

Isolation and Characterization of Podocytes from Mouse Models

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Isolation of podocytes from CD36 knockout mice (on C57BL6/J background, kindly provided by Dr. Deneys van der Westhuyzen at University Kentucky), TSP1 knockout mice (Jackson laboratory), or control wild type mice was performed as described previously [20 (link), 21 (link)]. Briefly, glomeruli were isolated from mice [21 (link)] and plated on collagen type I-coated dishes at 37°C in RPMI-1640 medium with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 mM HEPES, 1mM sodium bicarbonate, and 1mM sodium pyruvate to allow glomerular podocytes to grow out. Subculture of primary podocytes was performed by detaching the glomerular cells with 0.25% trypsin-EDTA, followed by sieving thought 40 µm cell strainer, and cultured on type 1 collagen coated dishes. Podocytes of passages 1 or 2 were used for the experiments.
CD36−/− podocytes or wild type podocytes were treated with 125 µM of palmitate (Sigma) or control BSA for 24 h. After treatment, cells were harvested. TSP1 expression in both mRNA levels and protein levels were determined by PCR and western blotting, respectively. TSP1−/− podocytes or wild type podocytes were treated with 125 µM of palmitate (Sigma) or control BSA for 24 hours. After treatment, caspase 3 activity was analyzed by using caspase-3 colorimetric assay kit (R&D). BSA filter assay was also analyzed in these cells.
Cui W., Maimaitiyiming H., Zhou Q., Norman H., Zhou C, & Wang S. (2015). Interaction of thrombospondin1 and CD36 contributes to obesity-associated podocytopathy. Biochimica et biophysica acta, 1852(7), 1323-1333.
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10

C3H10T1/2 Cell Proliferation and Apoptosis

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The proliferation of C3H10T1/2 cells was evaluated using an MTT Proliferation kit (Roche Applied Science). Cells were plated in 96-well plates at a density of 5×103 cells/well in 100 µl medium. After 24 h, cells were incubated with 10 µl MTT reagent for 2 h. Then, 100 µl of the solubilization solution from the kit was added to dissolve the formazan product. Absorbances at 570 nm were measured.
For the apoptosis assay, C3H10T1/2 cells were cultured under serum-starved conditions (DMEM-0.5% FCS) for 24 h. The proapoptotic activity was determined by assessing the caspase-3 activity using the Caspase-3 Colorimetric Assay kit (R&D Systems, Inc.) according to the manufacturer's protocol. Absorbances at 405 nm were read on a microplate reader.
Dong J., Xu X., Zhang Q., Yuan Z, & Tan B. (2021). Critical implication of the PTEN/PI3K/AKT pathway during BMP2-induced heterotopic ossification. Molecular Medicine Reports, 23(4), 254.
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