Click-iT proliferation assay with improved DNA histograms.

The Click-iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7-aminoactinomycin D (7-AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click-iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G(1) peak) and shorter processing time by eliminating the fixation and permeabilization steps.

ALDH(+)/CD44(+)/CD24(-) expression in cells from body cavity fluids.

BACKGROUND: Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers (CD44(+)/CD24(-)) in stem cells from breast tumors has been reported. This study was undertaken to monitor expression of these markers in cells from body cavity fluids of female patients suspected to have a malignancy. METHODS: Cells from peritoneal and pleural fluids of 100 female patients were examined by diagnostic cytology and analyzed by laser flow cytometry for enhanced ALDH1 expression. Cells from 36 body cavity fluids with ALDH1(bright) fluorescence were then analyzed for the expression of CD44 and CD24 markers. RESULTS: In samples positive for malignancy, ALDH1(bright) cells with both SSC(low) and SSC(high) were seen. In 15 body cavity fluids positive for malignancy, the percentage of ALDH1(bright) cells ranged from 0.26 to 6.34% of the total cells. The percentage of ALDH1(bright) cells with CD44(+)/CD24(-) expression in these samples ranged from 0.02 to 3.66%. ALDH1(bright) cells with CD44(+)/CD24(-) expression were also present in body cavity fluids of patients in whom diagnostic cytology could not detect any malignancy. However, the percentage of ALDH1(bright) and CD44(+)/CD24(-) cells amongst the 21 body cavity fluids with negative cytology was lower than that of samples with malignancy. CONCLUSIONS: Expression of ALDH1(bright) and the CD44(+)/CD24(-) phenotype in body cavity fluids in which diagnostic cytology could not find any malignant cells suggests that this phenotype may not be restricted to the putative breast tumor stem cells. It is possible that only subsets of cells with this phenotype are the putative breast tumor stem cells.

Click-iT assay with improved DNA distribution histograms.

The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2'-deoxyuridine analog, 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated "click" reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7-aminoactinomycin-D (7-AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CV's). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iT Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis.

Measurement of electronic volume versus DNA content of nuclei can be used to discriminate between normal and malignant cells. Epithelial membrane antigen immunocytochemistry (EMA-ICC), a helpful ancillary test in body cavity fluids, is not universally accurate for detecting malignancy in effusions. The current study was undertaken to determine if multiparametric flow cytometry (based on simultaneous analysis of light scatter, nuclear volume, DNA, and nuclear protein content) in combination with (EMA-ICC) could be used for the detection of malignant cells in peritoneal and pleural fluids. We studied 130 body cavity fluids (68 peritoneal and 62 pleural fluids) by conventional cytology and multiparametric laser flow cytometry. EMA-ICC was performed using EMA antibodies and L-SAB detection system (DakoCytomation, Carpinteria, CA). EMA-ICC had significantly higher sensitivity than conventional cytology (79% versus 59%, P = 0.016) and ploidy (79% versus 38%, P = 0.001). Cytology had significantly higher specificity than ploidy (97% versus 82%, P = 0.012). The differences in specificity between EMA-ICC and ploidy (87% versus 82%, P= 0.607) or EMA-ICC and cytology (87% versus 97%, P = 0.109) were not statistically significant. However, assuming serial testing, sensitivity increased significantly for the combinations of cytology and EMA-ICC (79.4%, P = 0.016) and cytology and ploidy (73.5%, P = 0.004) as compared to cytology alone (58.8%). Also, the combination of cytology and ploidy had a higher sensitivity than ploidy alone (73% versus 38%, P < 0.0001). However, the sensitivity associated with the three tests used in serial (85.3%) was not significantly different from the sensitivities corresponding to the combination of cytology and EMA-ICC (79%) or cytology and ploidy (73%). Multiparametric flow cytometry utilizing high resolution DNA, nuclear volume, protein measurement, and ICC, in combination with cytomorphology, may be a valuable tool for rapid identification of malignant cells in body cavity fluids.

Flow cytometric monitoring of fluorescent drug retention and efflux.

Laser flow cytometry has been used for monitoring cellular retention of fluorescent drugs such as fluorescent anticancer antibiotics (e.g., doxorubicin) and fluorochromes used for the detection of cellular drug efflux and resistance (e.g., rhodamine 123, Hoechst 33342). Multiparametric flow cytometry can be used for identification of tumor cell subpopulations based on their drug retention profiles with or without the presence of an efflux blocker. This rapid procedure can be used for identification of tumor cells with the drug-resistance phenotype based on drug efflux as well as for efflux blockers that may block efflux of a chemotherapeutic agent and thus increase cellular retention and sensitivity. It has been reported recently that some of the bone marrow stem cells (SP cells) efflux the Hoechst 33342 fluorochrome and thus can be rapidly identified by comparing red vs blue fluorescence in the presence or absence of an efflux blocker such as verapamil. The present chapter discusses some of the flow cytometric methods used for the study of cellular drug retention and the artifacts that may arise in such analysis.