Annexin 5 fluos kit

Caspase-3 activity was determined as an indicator of apoptosis. Detached cells were washed from plates with 500 μl PBS and collected by centrifugation. Adherent cells were lysed using a buffer containing 5 mM Tris HCl pH 7.4, 5 mM EDTA and 5% NP-40 and the lysate removed to the tube containing the corresponding cell pellet. Samples were frozen at –20°C until needed for processing. A 50 μl aliquot of the cell lysis was added to 1 ml of caspase substrate buffer containing 50 mM HEPES, 10% sucrose and 0.1% CHAPS with 10 mM DTT and 1 μl/ml of the caspase-3 fluorescent substrate Ac-DEVD-AFC (Calbiochem, EMD Millipore). Reactions were incubated at room temperature overnight prior to measurement of caspase activation using a spectrofluorimeter with excitation at 400 nm and emission at 505 nm. Apoptosis was also measured by Annexin-V and propidium iodide staining followed by flow cytometry using an Annexin-V FLUOS kit (Roche). Lactate dehydrogenase (LDH) release, as a measure of cytotoxicity, was determined using the Cytotox 96^® Non-Radioactive Cytotoxicity Kit (Promega, Madison, WI).

Fetal bovine serum (FBS; US origin) was purchased from HyClone Laboratories Inc., South Logan, UT, USA. Anti-caspase-3, anti-caspase-8, anti-PARP, anti-BID, anti-Bax, anti-COX-2 and anti-beta-actin antibodies were purchased from OriGene Technologies, Inc., Rockville, MD, USA. Anti-p53, anti-Bcl-2 (NT), anti-caspase-9 and anti-iNOS antibodies were purchased from AnaSpec, Inc., Fremont, CA, USA. Anti-Cdc25C, anti-CDK1, anti-CDK2, anti-Cyclin A1 and anti-Cyclin B1 antibodies were purchased from Bioss, Inc., Woburn, MA, USA. Anti-p21 (WAF1, Cip1) was purchased from eBioscience, San Diego, CA, USA. Anti-cyclin D1 and anti-cdk-4 antibodies were purchased from Cell Signaling Technology, Inc., Danvers, MA, USA. Cell Proliferation Reagent WST-1 and the Annexin-V-FLUOS Kit were purchased from Roche Diagnostics, Mannheim, Germany. Alkaline phosphatase-conjugated anti-Rabbit secondary antibody was purchased from RockLand Immunochemicals Inc., Gilbertsville, PA, USA. Z-IETD-FMK (caspase-8 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) were purchased from Abcam, Cambridge, MA, USA. All the other chemicals and solvents are of analytical or molecular biology grade and procured locally.

Cells were collected, washed by PBS and stained using the Annexin-V-FLUOS kit (Roche) according to manufacturer’s protocol. Stained cells were analyzed by flow cytometry on a CytoFLEX system (Beckman Coulter, Indianapolis IN, USA). The data were analyzed using Flowjo software (Three Star, Inc., Ashland, OR, USA).

Bioflavonoids and ethanolic crude extract from Citrus seeds were obtained from Ruiheng Industry Co., Limited., Hefei City, China. Dimethyl sulfoxide (DMSO), 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT dye), 2′,7′-dichlorohydrofluorescein diacetate (DCFH-DA), neohesperidin, hesperidin, naringin, and naringenin were obtained from Sigma-Aldrich, St. Louis, MO, USA. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum, penicillin G sodiumm and streptomycin were from GibcoBRL, Invitrogen Life Science Technologies, Thermo Fisher Scientific Inc., Waltham, MA, USA. Annexin V-Fluos kit and complete mini-protease inhibitor cocktail were obtained from Roche, Indianapolis, IN, USA. Caspase determining kits were obtained from Invitrogen Life Science Technologies, Thermo Fisher Scientific Inc., Waltham, MA, USA. Mouse monoclonal antibody to Bcl-2 associated X protein (Bax) and rabbit polyclonal to Bcl-xL, Bak, and Bid, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Abcam, Cambridge, UK. Super Signal West Pico Chemiluminescent Substrate was obtained from Thermo Fisher Scientific Inc. Waltham, MA, USA.

U-251 and U-87 cells were collected for detection of apoptosis using the Annexin-V-FLUOS Kit (Roche, U.S.A.) according to the manufacturer’s instructions. All the samples were assayed in triplicate.

Cells were treated with DCA (10 mM) for 48 hours and apoptosis was evaluated by using Annexin V Fluos kit (Roche) as per manufacturer’s instructions.

The indicated cells were collected for detection of apoptosis using the Annexin-V-FLUOS Kit (Roche, USA) according to the manufacturer’s instructions. All of the samples were assayed in triplicate.