Cell Cycle and Apoptosis Analysis

Cell cycle distribution including the pre-G₁ fraction was determined from DNA histograms as described before [54 (link)]. Apoptosis was quantified from detection of active, cleaved caspase-3 by flow cytometry using an Alexa Fluor^® 488-conjugated antibody (Cell Signaling Technology, Danvers, Massachusetts, USA) according to the manufacturer's protocol. Cell proliferation and the rate of DNA synthesis were determined by flow cytometry on AGS cells labelled with anti-BrdU antibody (AbD Serotec, Puchheim, Germany) and propidium iodide (PI, Sigma-Aldrich) according to the manufacturer's protocol. Briefly, cells were pulse labelled with 10 μM BrdU (Sigma-Aldrich) for 60 min, acid-treated and stained with an anti-BrdU and a secondary APC-conjugated antibody for determination of DNA synthesis and with 20 μg/ml PI for determination of total DNA content.

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Rohwer N., Bindel F., Grimm C., Lin S.J., Wappler J., Klinger B., Blüthgen N., Du Bois I., Schmeck B., Lehrach H., de Graauw M., Goncalves E., Saez-Rodriguez J., Tan P., Grabsch H.I., Prigione A., Kempa S, & Cramer T. (2015). Annexin A1 sustains tumor metabolism and cellular proliferation upon stable loss of HIF1A. Oncotarget, 7(6), 6693-6710.

Publication 2015

Alexa Fluor® 488-conjugated antibody anti-BrdU antibody BrdU

A dna Acid Alexa fluor 488 Anti antibody Antibody Apoptosis Brdu Caspase 3 Cell cycle Cell proliferation Cells Dna synthesis Flow cytometry Propidium iodide Pulse Synthesis

Corresponding Organization :

Other organizations : Charité - Universitätsmedizin Berlin, German Cancer Research Center, Max Delbrück Center, Max Planck Institute for Molecular Genetics, Duke-NUS Medical School, Maastricht University, Humboldt-Universität zu Berlin, Universities of Giessen and Marburg Lung Center, Centre for Human Drug Research, Leiden University, European Bioinformatics Institute, Wellcome Trust, RWTH Aachen University, Joint Research Centre, Universitätsklinikum Aachen

Top 5 similar protocols

Variable analysis

independent variables

Treatment/condition (e.g., drug treatment, control)

dependent variables

Cell cycle distribution including pre-G1 fraction
Apoptosis (detected by active, cleaved caspase-3)
Cell proliferation
Rate of DNA synthesis

control variables

Incubation time (60 min for BrdU labeling)

controls

Positive control: Detections of active, cleaved caspase-3 by flow cytometry using an Alexa Fluor® 488-conjugated antibody
Negative control: Not explicitly mentioned

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