Simultaneous evaluation of viability and Bcl-2 in small-cell lung cancer.
When designing molecular targeted therapeutic strategies against cancer, it is important to correlate protein expression and cell viability. However, such goal can be difficult if performed in separate assays, especially when only a fraction of cells has been efficiently transfected. Therefore, the aim of the present study was to establish a flow cytometry procedure to assess simultaneously Bcl-2 protein level and viability in small-cell lung cancer (SCLC) cells. Viability assessment was performed by staining cells with Annexin V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD). Intracellular detection of Bcl-2 was carried out by immunodetection with monoclonal antibodies. Regarding viability determination, the FSC/7-AAD plot identifies the same percentage of viable cells as the FSC/Annexin V-FITC plot, although with greater sensitivity. The procedures involving cells' fixation with 1% paraformaldehyde and permeabilization with digitonin, required for intracellular Bcl-2 immunostaining did not compromise the association of 7-ADD (nor Annexin V-FITC) previously incubated with SCLC cells. It was therefore possible to simultaneously assess cell viability and Bcl-2 protein in SCLC cells. A simple, sensitive, and versatile procedure was established for the first time for the simultaneous evaluation of cell viability and intracellular detection of Bcl-2 in SCLC.
Santos
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Faculty of Pharmacy, Laboratory of Pharmaceutical Technology, University of Coimbra, Coimbra, Portugal.
Sarmento-Ribeiro
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de Lima
M C Pedroso
MC
Simões
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Moreira
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JN
D017304:Annexin A5 / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology; D001549:Benzamides; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000276:immunology / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D000068877:Imatinib Mesylate; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D010879:Piperazines / Q000276:immunology / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011743:Pyrimidines / Q000276:immunology / Q000494:pharmacology; D055752:Small Cell Lung Carcinoma / Q000378:metabolism* / Q000473:pathology
D017304:Annexin A5; D000970:Antineoplastic Agents; D001549:Benzamides; D004791:Enzyme Inhibitors; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D011743:Pyrimidines; D005047:Etoposide; D000068877:Imatinib Mesylate