Computational protocol: Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex class I-related chain A and B in cancer cells

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Protocol publication

[…] Cancer cells were incubated for 24 h after exposure to 10 Gy X-rays, carbon ions, or aphidicolin treatment, and then harvested for FACS analysis. Adherent cells were harvested by shake-off in 2 mM EDTA/phosphate-buffered saline (PBS) without trypsinization according to the method described by Clayton et al (). Harvested cells were washed with FACS solution (ice-cold PBS containing 2% newborn calf serum, 1 mM EDTA, 0.01% w/v NaN3), and then stained with MICA/B antibodies for 20 min at 4°C. Cells undergoing apoptosis were detected using Annexin V. MICA/B expression was analyzed in cells doubly negative for propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, USA) and Annexin V (BioLegend, San Diego, CA, USA). FACS was performed on a FACSCalibur instrument using the CellQuest software. FACS data were analyzed using the FlowJo v. 9.3 software (Tree Star, Inc., Ashland, OR, USA). Expression levels of surface MICA/B were determined as mean fluorescence intensity (MFI) of anti-MICA/B normalized against the MFI of an isotype control antibody. The IR-induced fold increase in expression level was calculated by dividing the MFI of irradiated cells (IR-MFI) by the MFI of non-irradiated cells (non-IR-MFI). Reproducible results in all FACS experiments were obtained from two or more independent experiments. A representative FACS histogram is shown for each analysis. [...] Statistical significance in the FACS, cell proliferation and NK-mediated cytotoxicity assays were determined using Student's two-tailed t-test or the Mann-Whitney U test, both in SigmaPlot 12.0. […]

Pipeline specifications

Software tools FlowJo, SigmaPlot
Applications Miscellaneous, Flow cytometry
Diseases Neoplasms