Computational protocol: Role of stress-activated OCT4A in the cell fate decisions of embryonal carcinoma cells treated with etoposide

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Protocol publication

[…] Cells were harvested at relevant time points, washed in cold PBS and fixed with 70% ethanol for 20 min at room temperature. After two washes in tris buffered saline (TBS), cells were permeabilised with TBS/4% bovine serum albumin (BSA)/0.1% Triton X-100 for 10 min at room temperature. Samples were then incubated with mouse monoclonal anti-OCT4 antibody solution (Santa Cruz) and rabbit polyclonal p21Cip1 (Pierce) in TBS/4% BSA/0.1% Triton X-100 for 1h at room temperature. Following two washes in TBS, cells were incubated with goat anti-rabbit- IgG-Alexa Fluor 488 (A31627, Invitrogen) and Chicken anti-mouse IgG-Alexa Fluor 647 (A21463, Invitrogen) (1:200) in TBS/4% BSA/0.1% Triton × 100, for 30 min in the dark. DNA was counterstained with 10 μg/ml propidium iodide (PtdIns) in PBS, and assessed by flow cytometry using a FACS Aria (BD Biosciences) using Cell Quest Pro Software). Data was analyses using FlowJo analysis software. [...] Statistical analysis was performed in GraphPad (GraphPad Software Ltd). Student t-test was used to calculate the statistical significance of difference of means where appropriate. Statistical significance was accepted when p < 0.05. Graphs were plotted in GraphPad Prism 5 and IBM SPSS statistics 22. […]

Pipeline specifications

Software tools FlowJo, SPSS
Applications Miscellaneous, Flow cytometry
Diseases Carcinoma, Neoplasms, Brain Stem Neoplasms
Chemicals Etoposide