Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol

Glade, C.P.; Seegers, B.A.M.P.A.; Meulen, Etienne F. J.; Hooijdonk, C.A.E.M. van; Erp, P.E.J. van; Kerkhof, Peter C M van de

doi:10.1007/s004030050047

Cited by 18 publications

(40 citation statements)

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“…In order to obtain single-cell suspensions with a good separation of dermis and epidermis, we used an optimized thermolysin-trypsin protocol, as previously described in detail by Glade et al [12]. …”

Section: Methodsmentioning

confidence: 99%

“…To assess epidermal differentiation, the monoclonal antibody RKSE60 (Department of Molecular Biology, University of of Maastricht, The Netherlands) was used at a dilution of 1:20. This IgG1 type mouse monoclonal antibody stains K10, an intermediate filament-type protein that is only expressed in suprabasal keratinocytes [12, 13]. For measuring hyperproliferative cells, we used the IgG2a type mouse monoclonal antibody LHK6B (Novocastra Laboratories, Newcastle upon Tyne, UK).…”

Section: Methodsmentioning

confidence: 99%

“…TP3 intercalates with double-stranded regions of nucleic acids, and as it also affects RNA, it was used in combination with monoclonal goat antibodies against mouse IgG2a and IgG1, conjugated to fluorescein isothiocyanate (GAM-FITC, 1:50) and phyco-erythrin (GAM-PE, 1:250), respectively (Southern Biotechnology Associates, Birmingham, Ala., USA). The specificity and cross-reactivity of these second-stage reagents have been described before [12, 18]. …”

Section: Methodsmentioning

confidence: 99%

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Changes in Keratin 6 and Keratin 10 (Co-)Expression in Lesional and Symptomless Skin of Spreading Psoriasis

et al. 2000

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Background: Keratin 6 (K6) and keratin 10 (K10) are markers for epidermal hyperproliferation and differentiation, respectively, and are both expressed in the suprabasal layers of the epidermis. They may be co-expressed in different stages of the spreading psoriatic lesion, but single expression can also occur. Objective: To investigate to what extent keratinocytes express K6 and K10, and to what extent they co-express K6 and K10 in different stages of the psoriatic lesion. We studied this in spreading psoriatic plaques. Methods: Three 3-mm punch biopsies were obtained from the inner involved margin of a spreading lesion, from the uninvolved skin immediately adjacent to the spreading plaque, and from the distant uninvolved skin of 8 patients with incipient psoriasis. From 9 healthy volunteers, 3-mm punch biopsies were obtained as controls. After preparation of single cell suspensions of these biopsies, a triple staining protocol was performed with markers for K6 (monoclonal antibody LHK6B), K10 (monoclonal antibody RKSE60) and DNA content (TO-PRO-3 iodide). Subsequently, cells were measured with a flow cytometer and the proportion of the markers was calculated using specific software. Results: We observed a population of K6/K10-co-expressing cells, but also populations expressing only K6. These subpopulations varied with the involvement of the lesion. There was a statistically significant difference between the inner margin and the outer margin with respect to the proportion of K6- and K10-expressing cells, whereas more K6-positive and K10-negative cells were detected in the inner margin of the lesions. The proportion of K6/K10-co-expressing cells in the inner margin was significantly different from the distant uninvolved skin. Conclusion: We confirmed that individual keratinocytes in psoriasis can express K6 or K10 depending on their localization in involved or uninvolved skin. There is a unique subpopulation of cells in the psoriatic plaques which co-express K6 and K10. More studies are required to fully understand the pathogenic relevance of co-expression and single expression of K6 and K10.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Changes in Keratin 6 and Keratin 10 (Co-)Expression in Lesional and Symptomless Skin of Spreading Psoriasis

et al. 2000

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“…All treatments, except for the vehicle, resulted in higher percentages of normally differentiated, keratin-10-positive keratinocytes (reference value in normal skin 50–60% [11]). Values from patients treated with the combination product as well as betamethasone dipropionate were significantly higher than those of vehicle-treated patients.…”

Section: Discussionmentioning

confidence: 99%

“…The tissue was preserved in phosphate-buffered saline (PBS) until cell isolation, for a maximum of 8 h, as described previously [11]. In brief, the biopsies were kept in PBS (with calcium and magnesium) containing 0.5 mg/ml protease type x (Sigma, St. Louis, Mo., USA) at 4°C during 16–20 h. Subsequently, the dermis and epidermis were separated with a forceps, and the epidermis was incubated for 30 min at 37°C in PBS containing 0.025 mg/ml trypsin type III (Sigma) and 0.3 mg/ml dithioerythritol (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Treatment of Psoriasis with a New Combination of Calcipotriol and Betamethasone Dipropionate: A Flow Cytometric Study

Rossum¹,

Erp²,

Kerkhof³

2001

Dermatology

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Background: Calcipotriol and corticosteroids are established topical antipsoriatics. Previous studies have shown that combined therapy with calcipotriol and betamethasone dipropionate was more effective than monotherapy. In the present study, a recently developed combination product of calcipotriol and betamethasone dipropionate was compared with both monotherapies and the vehicle. Methods: Twenty-five psoriatic patients were treated twice daily with the combination product, monotherapy or vehicle during 4 weeks. Skin biopsies, taken before and after treatment, were analysed using a multi-parameter flow cytometric method. Parameters of inflammation (vimentin-positive cells), normal differentiation (keratin-10-positive cells) and proliferation (cells in SG₂M-phase) were assessed. Results: Flow cytometric analysis showed that the combination product turned out to be more effective in reducing inflammation compared with the other treatments. Restoration of normal differentiation was more advanced in patients treated with the combination product or betamethasone dipropionate compared to the vehicle. The highest number of normally differentiated cells was seen after use of the combination product. All treatments, except for the vehicle, decreased hyperproliferation. Conclusion: This study shows that the combination product is a valuable new approach to the treatment of psoriasis.

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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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Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol

Abstract: Multi para meter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol

Cited by 18 publications

References 2 publications

Changes in Keratin 6 and Keratin 10 (Co-)Expression in Lesional and Symptomless Skin of Spreading Psoriasis

Changes in Keratin 6 and Keratin 10 (Co-)Expression in Lesional and Symptomless Skin of Spreading Psoriasis

Treatment of Psoriasis with a New Combination of Calcipotriol and Betamethasone Dipropionate: A Flow Cytometric Study

Method for identifying prostate cells in semen using flow cytometry

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