The number of apoptotic cells was quantified using a Pacific Blue™ Annexin V apoptosis detection kit (BioLegend, San Diego, CA) according to the manufacturer's instructions. CCA cells were seeded into 12-well plate at 5×104 cells and treated with various concentrations of flavopiridol. The dead and adherent cells were harvested, incubated with Pacific Blue™ Annexin V at room temperature for 30 min in the dark and stained with 1 μg/ml propidium iodide (PI). The cells were analyzed using a BD LSR II™ flow cytometer (BD Bioscience, San Jose, CA). Data analysis was performed using FlowJo software (Tree Star Inc., Ashland, OR).
Pacific blue annexin 5 apoptosis detection kit
Manufactured by BioLegend
Sourced in United States
The Pacific Blue Annexin V Apoptosis Detection Kit is a laboratory product designed for the detection of apoptosis, a process of programmed cell death. The kit utilizes Pacific Blue-conjugated Annexin V, a protein that binds to phosphatidylserine, a lipid that becomes exposed on the surface of cells undergoing apoptosis. The kit provides a straightforward method for the identification and quantification of apoptotic cells through flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Lsr 2 flow cytometer, by BD (1 mentions)
Prism v7, by GraphPad (1 mentions)
Lsr 2 facs machine, by BD (1 mentions)
Apoptosis necrosis detection kit, by Abcam (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
Macsquant vyb, by Miltenyi Biotec (1 mentions)
Lsrii cytometer, by BD (1 mentions)
Accutase, by BioLegend (1 mentions)
7 protocols using pacific blue annexin 5 apoptosis detection kit
1
Quantifying Apoptosis in CCA Cells
2
Annexin V Apoptosis Assay
3
Quantifying Apoptosis by Microscopy and Flow Cytometry
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Apoptosis was evaluated by fluorescent microscope and flowcytometry, using an Apoptosis/Necrosis detection kit (Abcam) and Pacific Blue Annexin V Apoptosis Detection Kit (Biolegend), respectively. After appropriate treatments, cells were processed according to the manufacturer’s instructions. In microscopy experiments, a minimum of 1000 cells were counted per condition per experiment using Fiji software (http://fiji.sc/Fiji ). The data of flowcytometry were analysed with FlowJo software.
4
Apoptosis Analysis of PDAC Cells
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Cells were seeded in 6-well plates in duplicate (150 000 cells/2 ml media/well) and the day after, the corresponding treatment was administered. We treated four PDAC cells with 7 μM of perhexiline for 72 h, or for 24 h with perhexiline alone (7 μM) or in combination with gemcitabine (1 μM) for PDAC084T cells. After treatments, cells were detached with pre-warmed accutase (Gibco), resuspended in Annexin V-binding buffer, and stained for 30 min with the BioLegend’s Pacific Blue™ Annexin V Apoptosis Detection Kit with propidium iodide (PI), according to manufacturer’s instructions. Finally, ten thousand events per sample were acquired in a MACSQuant-VYB (Miltenyi Biotec) and data analysis was done with the FlowJo software.
5
Apoptosis and Necrosis Detection
6
Annexin V Apoptosis Detection
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Following Nilo1- or irrelevant control-treatment, the tumorspheres were dissociated with Accutase (5 min, 37°C), stained with Pacific Blue Annexin V Apoptosis detection kit (BioLegend) according to the manufacturer’s protocol, and analyzed by flow cytometry.
7
Apoptosis Detection by Flow Cytometry
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Apoptotic cells were detected using a Pacific Blue Annexin V apoptosis detection kit (BioLegend, San Diego, CA, USA) followed by surface marker staining and flow cytometry analysis. Early apoptotic cells were defined as annexin V‐positive and PI/7‐AAD‐negative. Late apoptotic cells were designated annexin V‐positive and PI/7‐AAD‐positive.
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