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

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‐associated 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% and from 2.71% to 4.71% for the DNA‐diploid and ‐aneuploid fractions, respectively. In DNA‐diploid tumors, antigen expression (HER‐2/Neu, proliferating cell nuclear antigen) could be analyzed without interference of fluorescence signals from nonneoplastic cells. Neoplastic tumor subpopulations were clearly identified based on both DNA‐ploidy status and heterogeneity of antigen expression. The present method offers new possibilities for multiparameter DNA FCM on clinical samples and enables the identification of intratumor neoplastic subpopulations based on antigen expression and DNA‐ploidy status. © 1996 Wiley‐Liss, Inc.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six‐color immunophenotyping

“…Several methods have been described for FC DNA‐ploidy analysis including methods that allow simultaneous evaluation of immunophenotyping and DNA‐ploidy . These methods use the dyes like propidium iodide (PI), 4′,6‐diamidino‐2‐phenylindole (DAPI), 7‐aminoactinomycin‐D (7‐AAD), DRAQ5, and Hoechst 33342 and these dyes are either excited with blue laser or red laser or UV laser.…”

High‐resolution multi‐parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin‐embedded tissues

“…A standard FACSCalibur (BD Biosciences, San Jose, CA, USA) was used for the simultaneous measurement of FITC, R‐PE, and PI as previously described 1, 15, 16. Twenty thousand keratin‐positive single cell events were collected using the FL3‐A versus FL3‐W pulse‐processor utility.…”

Heterogeneity of radiation induced apoptosis in Ewing Tumor cell lines characterized on a single cell level

“…A simultaneous characterization of light scatter properties, surface molecules, DNA content, and apoptosis-associated markers can be performed at a single cell level. [11][12][13][14] As an early marker of apoptosis the binding of Annexin V to phosphatidylserine on the cell membrane can be used. 15 In addition, the staining of the DNA with dyes like 7-AAD without any permeabilisation of the cell membrane allows the characterization of the integrity of the cell membrane and is therefore a useful marker for characterization of apoptosis.…”