Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis

Doornbos, Richard; Grooth, B.G. de; Kraan, Yvonne M.; Poel, C.J. van der; Greve, Jan

doi:10.1002/cyto.990150312

Cited by 34 publications

(25 citation statements)

References 8 publications

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“…Excitation of this dye is accomplished with the 635-nm laser and can be detected in a 661 ± 8 nm bandpass detector without further compensation in the fluorescein isothiocyanate (FITC) and PE detectors. To-Pro 3 has been used in three-color flow cytometry in the testing of skin melanoma [20][21][22]. Using the antibodies to PSMA and cytokeratin, as well as the DNA stain To-Pro 3, we were able to identify viable prostate cells in semen in normal as well as prostate cancer patients.…”

Section: Methodsmentioning

confidence: 99%

Method for identifying prostate cells in semen using flow cytometry

Barren¹,

Holmes²,

Boynton³

et al. 1998

Prostate

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BACKGROUND Prostate‐specific antigen (PSA) cannot differentiate benign prostatic hyperplasia (BPH), from prostatitis, or prostate cancer in the range of 4.0–10 ng/ml. An accurate cytologic or histologic assessment is necessary to confirm the proper diagnosis. The nature of a biopsy tends to make it a selective test not frequently repeated. We are reporting a technique employing semen as a source for the differential diagnosis of prostate epithelial cells. METHODS Eleven vasectomized and nonvasectomized prostate cancer patients provided semen samples (stage T1 to T2). Two patients provided repeat samples. In addition, 15 vasectomized or nonvasectomized individuals without evidence of disease provided semen samples. Three million cells fixed with 50% ethanol were stained by an antibody (7E11.C5) to prostate‐specific membrane antigen (PSMA), Hybritech Antibody (399) to PSA, and cytokeratin 8 and 18. In addition to the antibodies described, a DNA stain To‐Pro 3 was used to identify 2n‐4n DNA containing cells. A dual laser, Becton Dickinson FACSCaliber cytometer, was used to analyze the samples. RESULTS All semen specimens contained diploid, cytokeratin 18–positive epithelial cells regardless of disease status. A clear difference between prostate cancer and normal prostate cell samples was observed using staining with 7E11.C5. The ratio of prostatic cells in the total epithelial cell population (PSMA:cytokeratin ratios) was calculated for each specimen. A retrospective study of sixteen semen samples from 11 prostate cancer patients had a mean PSMA:cytokeratin ratio of 0.57, whereas the samples from 15 patients without evidence of cancer had a mean PSMA:cytokeratin ratio of 0.11. This difference was significant. PSA staining was variable and inconsistent. CONCLUSIONS This report demonstrates that human semen contains prostate cells that can be characterized and used in the clinical diagnosis of prostate cancer. Prostate 36:181–188, 1998. © 1998 Wiley‐Liss, Inc.

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Section: Methodsmentioning

confidence: 99%

Method for identifying prostate cells in semen using flow cytometry

Barren¹,

Holmes²,

Boynton³

et al. 1998

Prostate

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“…So others have chosen to use a dual-beam UV and 488 nm system and have used Hoechst 33342 as the DNA stain (1,141. Nevertheless, having a t our disposal an inexpensive low-power air-cooled HeNe laser (633 nm), we searched for a new red emitting fluorophore which could be excited with this laser. Recently, a family of new DNA probes with exceptional spectral properties has become available from Molecular Probes, Inc. (Eugene, OR) (7,9). TP3, which has peak absorbance at 642 nm and emission at 661 nm, is best excited by the HeNe laser at 633 nm.…”

mentioning

confidence: 99%

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

1994

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A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper.A subequilibrium concentration of 1 pM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In humanand mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation.Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. 0 1994 Wiley-Liss, Inc.

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“…and TO-PRO-3 (20) absorb at 642 nm and therefore are suitable for excitation with a 635-nm diode laser or the red line of the He-Ne laser [54,55]. TO-PRO-3 proved to be an excellent alternative to propidium iodide for measurement of the DNA content using flow cytometry.…”

Section: Intercalating Probesmentioning

confidence: 99%