Rapamycin's Impact on Oral Cancer Cells

As described by our previous studies (13 (link), 14 (link), 16 (link)), to evaluate the effect of rapamycin on damage to oral cancer cells, a H2A.X flow cytometry was performed. In addition, after treating Ca9-22 cells with the studied concentrations of rapamycin, they were trypsinized and then fixed with 75% ethanol for 15 min. The centrifugation of the samples was carried out to eliminate the fixative solution. Afterward, a permeabilization solution containing 1% BSA/0.2% Triton/1X PBS was added to the cells and they were then incubated in the dark at 4°C overnight with the first phospho-histone H2A.X (Ser139) monoclonal antibody from Santa Cruz Biotechnology at a dilution 1/100, washed twice with PBS and incubated with the secondary antibody conjugated to Alexa Fluor 488 from Santa Cruz Biotechnology in a 1:100 ratio for 1h before analyzing them with the BD flow cytometry system (BD FACS Canto II) and the percentage of positive cells was calculated. This experiment was repeated three times.

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Semlali A., Papadakos S., Contant C., Zouaoui I, & Rouabhia M. (2022). Rapamycin inhibits oral cancer cell growth by promoting oxidative stress and suppressing ERK1/2, NF-κB and beta-catenin pathways. Frontiers in Oncology, 12, 873447.

Publication 2022

Alexa Fluor 488 BD FACS Canto II

Alexa fluor 488 Antibody Centrifugation Dilution Ethanol Fixative Flow cytometry Histone h2a x Monoclonal antibody Oral cancer Rapamycin

Corresponding Organization :

Other organizations : Université Laval

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Variable analysis

independent variables

Rapamycin concentrations

dependent variables

Percentage of cells positive for phospho-histone H2A.X (Ser139)

control variables

Ca9-22 cell line
Trypsinization and fixation with 75% ethanol for 15 min
Permeabilization solution containing 1% BSA/0.2% Triton/1X PBS
Incubation with phospho-histone H2A.X (Ser139) monoclonal antibody (1/100 dilution) overnight at 4°C
Incubation with secondary antibody conjugated to Alexa Fluor 488 (1:100 ratio) for 1h
Analysis with BD flow cytometry system (BD FACS Canto II)

controls

No positive or negative controls were explicitly mentioned in the protocol.

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