One-tube triple staining method for flow cytometric analysis of DNA ploidy and phenotypic heterogeneity of human solid tumors using single laser excitation

Cytometry
W E CorverG J Fleuren

Abstract

We have developed a "one-tube" triple staining procedure that allows the identification of intratumor phenotypic subpopulations by FCM. Solid tumors were dissociated by a combined mechanical/ enzymatic method. Ovarian ascites tumor cell aggregates were enzymatically dissociated using trypsin. An antikeratin 8/18 MAb was used to label the epithelial fraction of these tumor samples. A second MAb directed against the leukocyte common antigen (LCA) was applied to identify nonneoplastic DNA-diploid cells. Other MAbs used as a second marker were directed against a tumor-associate surface, a cytoplasmic, or a nuclear antigen. Cells were stained using subclass-specific fluorescein-isothiocyanate (FITC) or R-phycoerythrin (PE)-conjugated antibodies. DNA was stained with propidium iodide (PI). Triply stained samples were measured on a standard bench-top flow cytometer (FACScan). Keratin 8/18-positive cells, LCA-positive cells, and DNA could be simultaneously detected in dissociated breast carcinomas, mixed Müllerian tumors, and ovarian ascites specimen for refining DNA index (DI) calculations and S phase fraction (SPF) determination. Coefficients of variation (CV) of the G0G1 peak of the DNA histograms obtained ranged from 2.55% to 4.64%...Continue Reading

Citations

Dec 17, 2015·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Prashant TembhareP G Subramanian
Jun 24, 2003·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·M PlanderR Knuechel
Feb 13, 2010·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Arabel Vollmann-ZwerenzGero Brockhoff
Feb 16, 2005·Apoptosis : an International Journal on Programmed Cell Death·S KönemannA Schuck
Feb 16, 2013·British Journal of Cancer·J H S DayalA M Thompson

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