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Caspaseglo kit

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The CaspaseGlo kit is a luminescent assay used to measure caspase-3/7 activity in cell-based assays. It utilizes a luminogenic caspase-3/7 substrate, which generates a luminescent signal proportional to the amount of caspase-3/7 activity present in the sample.

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Lab products found in correlation

3096 luminometer, by BD (3 mentions) Anti p akt, by Cell Signaling Technology (1 mentions) Anti pax7 antibody, by Developmental Studies Hybridoma Bank (1 mentions) Apoptag kit, by Merck Group (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) C2c12 cell, by American Type Culture Collection (1 mentions) Anti parp, by Cell Signaling Technology (1 mentions) Cetuximab, by Promega (1 mentions) Glomax multi detection system, by Promega (1 mentions) Pierce 660 assay, by Thermo Fisher Scientific (1 mentions) Glomax 20 20 luminometer, by Promega (1 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Fty720, by Selleck Chemicals (1 mentions) Luminoskan ascent, by Thermo Fisher Scientific (1 mentions) Cytation 5, by Agilent Technologies (1 mentions) Z vad fmk, by Promega (1 mentions) Z lehd fmk, by Promega (1 mentions) Z ietd fmk, by Promega (1 mentions) Macsquant analyzer 10 cytometer, by Miltenyi Biotec (1 mentions) Cyquant reagent, by Thermo Fisher Scientific (1 mentions)

20 protocols using caspaseglo kit

1

Isolation and Characterization of Primary Muscle Cells

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Primary muscle cultures (pp6) were isolated as described (Qu et al., 1998 (link)) from the indicated mouse lines. FoxO3-depleted (FoxO-KO) pp6 cells were prepared from foxO3+/− mice as previously described (Dentice et al., 2010 (link)). C2C12 cells were obtained from ATCC. In some experiments, endogenous T3 and T4 were removed from the FBS by charcoal absorption (Larsen, 1972 (link)). Transient transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Single myofibers were prepared from the extensor digitorumlongus and gastrocnemius muscles of 6- to 12-week-old mice as previously described (Rosenblatt et al., 1995 (link)). Pax7Hi-Lo isolation by FACS has been described elsewhere (Rocheteau et al., 2012 (link)). Anti-MyoD (sc-304), myogenin (sc-12732), tubulin (sc-8035), and anti-FoxO3 antibodies were purchased from Santa Cruz Biotechnology. Polyclonal anti-MHC antibody (MF-20a) and anti-Pax7 antibody were from Developmental Studies Hybridoma Bank. Anti-D3 antibody is described elsewhere (Huang et al., 2003 (link)). Anti-Desmin antibody was from MP Biomedicals (#10519). Anti-total Akt was from Upstate, and anti-pAkt and anti-PARP were from Cell Signaling Technology. TUNEL assay was performed using the ApopTag kit from Millipore. Caspase-3/Caspase-7 activity was measured by using the CaspaseGlo kit from Promega.
Dentice M., Ambrosio R., Damiano V., Sibilio A., Luongo C., Guardiola O., Yennek S., Zordan P., Minchiotti G., Colao A., Marsili A., Brunelli S., Del Vecchio L., Larsen P.R., Tajbakhsh S, & Salvatore D. (2014). Intracellular Inactivation of Thyroid Hormone Is a Survival Mechanism for Muscle Stem Cell Proliferation and Lineage Progression. Cell Metabolism, 20(6), 1038-1048.
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2

Apoptosis Induction in ATII Cells

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ATII cells were collected after a 24 h treatment with bleomycin (Bleo, 50 mU/mL), dexamethasone (Dex, 100 nmol/L), a combination of bleomycin and dexamethasone (Bleo + Dex) or vehicle (Ctl, saline 0,9%) and then caspase‐3/7 activity was determined by luminescent assay (630/595 nm) using the Caspase Glo kit (Promega), following the manufacturer instructions.
Aubin Vega M., Chupin C., Pascariu M., Privé A., Dagenais A., Berthiaume Y, & Brochiero E. (2019). Dexamethasone fails to improve bleomycin‐induced acute lung injury in mice. Physiological Reports, 7(21), e14253.
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3

Apoptosis Assay for Glioma and Cancer Cells

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VUGX01 (VU0007775) was prepared at 20mM in DMSO. Glioma cells (C6), colorectal cancer lines (Difi, DLD1, HCT116), and human embryonic kidney cells Hek293, were each plated in multiple wells (1×104 cells per well) of 96-well plates. 24 hours after the seeding, VUGX01 or Cetuximab (purchased from Vanderbilt Hospital Pharmacy) was added into the wells of the plates for a 24 hours treatment, then apoptosis assays based on Caspase 3/7 activity were performed (caspase-Glo kit, Promega, Madison, WI) according to manufacturer-recommended procedures.
Xie J, & Gore J.C. (2020). The Potential Targets and Mechanisms of a Carbazole and Pyrazole Containing Anticancer Compound. Current cancer drug targets, 20(5), 364-371.
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4

Caspase 3/7 Activity Assay

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Cells were seeded at 1.0 × 105 cells in a 96-well plate. After the culture for 24 h, the test compounds were treated for 17 h. The caspase 3/7 activity was measured using the Caspase Glo kit (Promega, WI, USA) according to the manufacturer’s instructions. Cells were incubated with 100 μL of Caspase Glo reagent at room temperature for 60 min. Following the caspase cleavage, the substrate reacts with luciferase and releases luminescent light in the presence of ATP and oxygen. The luminescence of the reaction products was measured using a microplate reader (GLOMAX MULTI Detection system, Promega, WI, USA).
Taira J., Sonamoto M, & Uehara M. (2017). Dual Biological Functions of a Cytoprotective Effect and Apoptosis Induction by Bioavailable Marine Carotenoid Fucoxanthinol through Modulation of the Nrf2 Activation in RAW264.7 Macrophage Cells. Marine Drugs, 15(10), 305.
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5

Cell Viability and Apoptosis Assays

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Cell viability was determined by MTT assay as described previously (21 (link)). Long-term viability was determined by performing Clonogenic assays. Cell cycle analysis was performed by staining the cells with Telford reagent as described previously (22 (link)). Caspase 3/7 activity was determined by Promega Caspase-Glo kit (Madison, WI, USA) as described previously (23 (link), 24 ).
Tadros S.B., Shukla S.K., King R.J., Gunda V., Vernucci E., Abrego J., Chaika N.V., Yu F., Lazenby A.J., Berim L., Grem J., Sasson A.R, & Singh P.K. (2017). De Novo Lipid Synthesis Facilitates Gemcitabine Resistance Through Endoplasmic Reticulum Stress in Pancreatic Cancer. Cancer research, 77(20), 5503-5517.
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6

Caspase Activity Measurement Protocol

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Activities of caspase-3/7 and -9 were measured in CCLR buffer with Caspase-Glo® kit (Promega, Madison, WI, USA; Caspase-3/7 –G811A, Caspase-9 –G8211) as described before [28 (link)]. Protein concentrations were measured with Pierce 660 assay (Thermo Fisher Scientific, 22660). Samples were introduced in a caspase-activity reagent mix to measure luminescence by GloMax® 20/20 Luminometer (Promega, E5311).
Pirc Marolt T., Kramar B., Bulc Rozman K., Šuput D, & Milisav I. (2020). Aripiprazole reduces liver cell division. PLoS ONE, 15(10), e0240754.
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7

Caspase 3/7 Activity Measurement

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Cells were seeded in a 96-well plate at a density of 3000 cells/well. Cell death was induced, and the Caspase 3/7 activity was measured at 24h and 48h using the caspase-glo kit (Promega #G8091). A fold change to untreated cells was taken to obtain caspase 3/7 activity.
Sprooten J., Vanmeerbeek I., Datsi A., Govaerts J., Naulaerts S., Laureano R.S., Borràs D.M., Calvet A., Malviya V., Kuballa M., Felsberg J., Sabel M.C., Rapp M., Knobbe-Thomsen C., Liu P., Zhao L., Kepp O., Boon L., Tejpar S., Borst J., Kroemer G., Schlenner S., De Vleeschouwer S., Sorg R.V, & Garg A.D. (2024). Lymph node and tumor-associated PD-L1+ macrophages antagonize dendritic cell vaccines by suppressing CD8+ T cells. Cell Reports Medicine, 5(1), 101377.
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8

Annexin V and Caspase 3/7 Assay

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For the Annexin V assay, sub-confluent cells were cultured in 6-well plates and harvested by trypsinization. Cells were washed twice with cold PBS and then resuspended in binding buffer. Annexin V and propidium iodide (PI) staining were performed according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA), and the Annexin V and PI signals were measured by FACS. To detect caspase 3/7 activities, the cells were cultured in 96-well plates at a density of 1×104 per well. After 24 h of treatment, the caspase 3/7 activity was determined by a Caspase-Glo kit (Promega). All of the experiments were performed in triplicate.
Tu B., Zhu J., Liu S., Wang L., Fan Q., Hao Y., Fan C, & Tang T.T. (2016). Mesenchymal stem cells promote osteosarcoma cell survival and drug resistance through activation of STAT3. Oncotarget, 7(30), 48296-48308.
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9

Caspase-Mediated Apoptosis Assay

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FTY720 and DTX were purchased from Selleck Chemicals (Houston, USA) and LC laboratories (Woburn, USA), respectively. Caspase-GLO kit was obtained from Promega (Fitchburg, USA). Other reagents and chemicals used were purchased from Sigma-Aldrich (Dorset, UK) unless otherwise specified.
Alshaker H., Wang Q., Srivats S., Chao Y., Cooper C, & Pchejetski D. (2017). New FTY720-docetaxel nanoparticle therapy overcomes FTY720-induced lymphopenia and inhibits metastatic breast tumour growth. Breast Cancer Research and Treatment, 165(3), 531-543.
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10

Quantitative Caspase Activation Assay

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For caspase activation assays, cells were plated at a density of 104 cells/well in opaque 96-well plates 24 hours prior to treatment. Twenty-four hours after treatment, activation of effector caspases 3 and 7 was evaluated with Promega’s CaspaseGlo kit (G8090) following manufacturer’s instructions. Resulting mixtures were quantified after 30 minutes of incubation at room temperature in a Beckton Dickinson BD 3096 luminometer.
Serrano-Oviedo L., Ortega-Muelas M., García-Cano J., Valero M.L., Cimas F.J., Pascual-Serra R., Fernandez-Aroca D.M., Roche O., Ruiz-Hidalgo M.J., Belandia B., Giménez-Bachs J.M., Salinas A.S, & Sanchez-Prieto R. (2018). Autophagic cell death associated to Sorafenib in renal cell carcinoma is mediated through Akt inhibition in an ERK1/2 independent fashion. PLoS ONE, 13(7), e0200878.
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