BITC-induced apoptosis was measured based on (a) the activation of caspase 3/7 and (b) the increase in DNA fragmentation. The activation of caspase-3/7 was assessed using the substrate (Z-DEVD)2-R110 (Bachem, Torrance, CA, USA) as described previously [54 (link)]. Briefly, cells were plated in 96-well plates and allowed to attach during overnight incubation. The cells were pretreated for 2 hrs with the indicated inhibitor and then treated with DMSO (control) or the indicated concentrations of BITC. Subsequently, the cells were directly lysed by adding caspase 3/7 assay buffer containing (Z-DEVD)2-R110 substrate, and the lysates were incubated at 37°C for 1 hr. The fluorescence intensity of the proteolytically released fluorophore R110 was then measured using a plate reader (Victor X2, PerkinElmer, Inc., Waltham, MA, USA) at an excitation wavelength of 485 nm and emission wavelength of 535 nm. The induction of DNA fragmentation in BITC-treated cells was detected using a BD Pharmingen™ APO-DIRECT™ Kit (Becton Dickinson, BD; NJ, USA) according to the manufacturer's instructions. DNA fragmentation was detected using a FACSCalibur flow cytometer, and the data were analyzed using CellQuest Pro software (BD, NJ, USA).
Z devd 2 r110
Manufactured by Bachem
Sourced in United States
Z-DEVD)2-R110 is a fluorogenic substrate used for the detection and quantification of caspase-3 and caspase-7 activity in biological samples. It is a peptide-based compound that contains the caspase-3/7 recognition sequence (DEVD) and is conjugated to a fluorescent dye (rhodamine 110).
Automatically generated - may contain errors
Lab products found in correlation
Victor x2, by PerkinElmer (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
3 amino 1 2 4 triazole 3 at, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
4 protocols using z devd 2 r110
1
Apoptosis Assays for BITC-Treated Cells
2
Caspase-3/7 Activity Assay Protocol
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The activation of caspase-3/7 was determined using the substrate (Z-DEVD)2-R110 (Bachem, Torrance, CA, USA) as described previously37 . Briefly, cell viability in cells treated with indicated concentrations of cisplatin for 24 hrs with or without AO-PDT treatment was measured using WST-1 reagent as described above. Subsequently, the caspase 3/7 activity was detected in the cells by adding assay buffer containing (Z-DEVD)2-R110 substrate and incubated at 37 °C for 1 hr. The fluorescence intensity of the proteolytically released fluorophore R110 was then measured using a plate reader (Victor X2) at an excitation wavelength of 485 nm and emission wavelength of 535 nm.
3
Caspase-3 Activity Measurement Assay
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Caspase-3 activity was assayed using (H-Asp-Glu-Val-Asp)2-Rhodamine 110 ((Z-DEVD)2-R110, Bachem, Torrance, CA, USA) substrate as previously described.17 (link),18 (link) In brief, cells were seeded in 96-well plates and incubated overnight. The cells were then treated with DMSO (control) or 1 µM RAD001 with or without the 2 hours pretreatment of inhibitors (1 mM 3-MA or 200 nM Baf A1) for 48 hours. The cells were subsequently lysed with caspase-3 assay buffer containing (Z-DEVD)2-R110 substrates and incubated at 37°C for 1 hour. The fluorescent intensity of proteolytically released fluorophore R110 was then measured using a plate reader (Victor X2; PerkinElmer, Waltham, MA, USA) with an excitation wavelength of 485 nm and emission wavelength of 535 nm.
4
Prostate Cancer Cell Lines and BITC Treatment
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The human prostate cancer cell lines CWR22Rv1 (Rv1) and PC3 were obtained from the American Type Culture Collection (ATCC) and maintained as described previously [53 (link)]. Cell culture reagents including RPMI 1640 medium, a penicillin and streptomycin antibiotic mixture, fetal bovine serum, non-essential amino acids, sodium pyruvate and glutamine supplements were obtained from Invitrogen (Carlsbad, CA, USA). BITC (purity of ~98%) was purchased from Sigma (St. Louis, MO, USA). The stock solution of BITC was prepared at a concentration of 10 mM in DMSO, and aliquots were stored at -20°C. Each cell line was cultured at 37°C in an atmosphere containing 5% CO2. The cells were treated with the indicated concentrations of BITC, and the control cells received an equal volume of DMSO. The final concentration of DMSO was less than 0.1%. The catalase, catalase inhibitor (3-Amino-1,2,4-triazole, 3-AT), 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), propidium iodide (PI), and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Sigma. The caspase 3 substrates (Z-DEVD)2-R110 were purchased from Bachem (Torrance, CA, USA). All other chemicals were purchased from Sigma unless otherwise specified.
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