PNC-mediated apoptotic outcomes on MDA-MB-231 cells were assayed by the Vybrant Apoptosis Assay Kit #2 (Invitrogen). After treatment with PNC (0, 62.5, 125, and 250 μM) for 48 h, MDA-MB-231 cells were rinsed twice using sterile PBS after which they were pelleted. Supernatants remained unused and were discarded while cells resuspended in 1X Annexin-binding buffer. To evaluate early apoptosis, cell staining was done using propidium iodide and Alexa Fluor 488 Annexin V using the Vybrant Apoptosis Assay Kit #2 (Invitrogen). Fluorescence-activated cell sorting (FACS) was carried out by FACScan flow cytometry (Becton-Dickinson, Sunnyvale, CA, United States). Data were obtained using the CELL Quest software.
Vybrant apoptosis assay kit 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vybrant Apoptosis Assay Kit #2 is a fluorescence-based kit used to detect and measure apoptosis, a programmed cell death process, in cell populations. The kit provides the necessary reagents to perform a quantitative analysis of apoptotic cells.
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Lab products found in correlation
Facscalibur, by BD (4 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Celltiter blue cell viability assay, by Promega (2 mentions)
Rnase a, by Merck Group (2 mentions)
Caspase glo 3 7 assay, by Promega (2 mentions)
Cellquest, by BD (2 mentions)
Cellquest pro software, by BD (2 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Tartrate resistant acid phosphatase staining kit, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Rankl, by R&D Systems (1 mentions)
Fc500 cytometer, by Beckman Coulter (1 mentions)
Zoe fluorescent cell imager, by Bio-Rad (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 assay, by Dojindo Laboratories (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
25 protocols using vybrant apoptosis assay kit 2
1
Apoptosis Evaluation in MDA-MB-231 Cells
2
Osteoclast Differentiation Assay
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DHA was purchased from Sigma-Aldrich (St Louis, MO, USA). The Alpha modification (α-MEM), dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco-BRL (Gaithersburg, MD, USA). The cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Tokyo, Japan). Soluble mouse macrophage colony-stimulating factor (M-CSF) and RANKL were obtained from R&D Systems (USA). The specific primary antibodies and secondary antibodies used in the experiments were obtained from Cell Signaling Technology (Cambridge, MA, USA) and Sigma-Aldrich (St Louis, MO, USA). The tartrate-resistant acid phosphatase (TRAP) staining kit, Triton X-100, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. The Vybrant® Apoptosis Assay kit #2 was from Invitrogen (Carlsbad, CA, USA).
3
Apoptosis detection by flow cytometry
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Cells were seeded at 200,000 cells per well in six-well plates. NICD-1 plasmid transfection cells were harvested at 24 hours after 5 μg/ml cisplatin treatment and 36 hours for DAPT treatment cells. Cells were washed twice with cold PBS and then re-centrifuged the washed cells, discarded the supernatants and resuspended the cells in 1X annexin-binding buffer. Early apoptosis was detected by staining with Alexa Fluor® 488 annexin V and Propidium iodide labeling using the Vybrant® Apoptosis Assay Kit #2 (Invitrogen, USA). FACS was performed using a FACScan flowcytometer (Beckman-Altra, USA). Data were acquired using CELL Quest software.
4
Cytotoxicity Assessment of Cold Atmospheric Plasma
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Cells were treated with Alexa Fluor® 488 annexin V/ propidium iodide (Vybrant® Apoptosis Assay Kit #2; Invitrogen) according to the manufacturer’s instructions 24 h after CAP application in order to determine the cytotoxicity of CAP by flow cytometric analysis with the Cytometer FC500 (Beckman coulter, Brea, CA, USA). STS (Sigma-Aldrich; 10 nM) treated cells served as positive control. Additionally, in a separate experimental set, controls and CAP-treated cells were also stained with the above kit components and subsequently the immunofluorescence was analyzed with the ZOE™ Fluorescent Cell Imager (Bio-Rad) at 1 h, 4 and 24 h after treatment.
5
Evaluating Anti-Inflammatory and Anti-Apoptotic Effects
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AP and PMA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Minimum Essential Medium-α (α-MEM), fetal bovine serum (FBS) and penicillin were obtained from Gibco-BRL (Gaithersburg, MD, USA). The Cell Counting kit (CCK)-8 assay was purchased from Dojindo Molecular Technology (Tokyo, Japan). Primary antibodies (monoclonal rabbit antibody; species reactivity, human) for β-actin, phospho-IκBα, IκBα and MMP-9 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The Luciferase Assay system was from Promega (Sydney, Australia). Tris, glycine, NaCl, SDS, and other reagents were from Sigma-Aldrich. The Vybrant® Apoptosis Assay kit #2 was from Invitrogen (Carlsbad, CA, USA).
6
Apoptosis Assay of HP on Breast Cancer Cells
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The apoptotic effects of HP on MDA-MB-231 and MCF-7 cells were determined using the Vybrant apoptosis assay kit #2 (Invitrogen, USA). Cells were treated with increasing concentrations of HP for 48 h. Then, they were washed twice with cold PBS and pelleted; the supernatants were discarded, and the cells resuspended in annexin binding buffer. Early apoptosis was detected by staining with Alexa Fluor 488 annexin V and propidium iodide. Fluorescence-activated cell sorting (FACS) was performed using a FACScan flow cytometer (Becton-Dickinson, Sunnyvale, CA, USA). Data were acquired using CELL Quest software.
7
Quantifying TQ-induced Apoptosis in SAOS3 Cells
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TQ-induced apoptotic SASVO3 death was quantified by flow cytometry with annexin V-FITC and PI staining. In brief, the cells were treated with TQ for 24 h. The treated cells were collected, washed twice with PBS, and subjected to annexin V and PI staining by using Vybrant Apoptosis Assay Kit 2 (Invitrogen, Carlsbad, CA) according to the manufacturer's step-by-step protocol. Recombinant annexin V conjugated to fluorophores and Alexa fluoro 488 dye provided maximum detection sensitivity. After staining, flow cytometry was performed to quantify apoptotic cells [20] (link).
8
Apoptosis and Cell Cycle Analysis of Breast Cancer Cells Treated with EGCG and SFN
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Precancerous SH cells and early transformed breast cancer SHR cells treated with either EGCG at 20 μM or SFN at 10 μM alone or together were collected and washed with cold phosphate-buffered saline (PBS). Cellular apoptosis was analyzed by the Vybrant Apoptosis Assay kit #2 (Invitrogen) as reported previously [23 (link)]. PI staining-based flow cytometry cell cycle assay was used to analyze cell cycle distribution. After washing with PBS, cells were fixed in 70% ethanol at -20°C overnight and washed with PBS twice. Cells were then suspended in PBS containing 0.1% Triton X-100, 0.1% RNase and 50 μg/ml PI and incubated in dark for about 30 min. Flow cytometry was used for both cell apoptosis and cell cycle analyses on a Becton Dickinson FACSCalibur Flow Cytometer. The fluorescence intensity of the viable cells was analyzed using CellQuest software.
9
Overexpression of lncRNA GAS5 induces apoptosis
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A375 overexpressed with pCDNA3.1-lncRNA GAS5 or vector control was plated in 6-well plates. After 48 h incubation, the cells were collected and subjected to Annexin V and propidium iodide staining by using Vybrant Apoptosis Assay Kit 2 (Invitrogen) according to the manufacturer's protocol. After staining, flow cytometry was performed to quantify apoptotic cells.
10
Quantification of Apoptosis by Flow Cytometry
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Apoptosis was quantified by flow cytometry with annexin V-FITC and PI staining. In brief, the cells were treated with SB alone or combined cisplatin treatment for 24 h. The treated cells were collected, washed twice with PBS, and subjected to annexin V and PI staining by using Vybrant Apoptosis Assay Kit 2 (Invitrogen, Carlsbad, CA) according to the manufacturer's step-by-step protocol. Recombinant annexin V conjugated to fluorophores and Alexa fluoro 488 dye provided maximum detection sensitivity. After staining, flow cytometry was performed to quantify apoptotic cells
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