Cytometry最新文献

Keratins are a group of cytoskeletal proteins that are found in human epidermis and other stratified squamous epithelia. Several different types of keratins have been described. Keratin 10 (K10) is a keratin that is expressed in well differentiated, suprabasal keratinocytes, and keratin 6 (K6) is a keratin which is associated with hyperproliferation. Psoriasis is a chronic inflammatory skin disease, and besides inflammation, disturbed differentiation and hyperproliferation are its hallmarks. In order to study the hyperproliferation associated keratinization in both well differentiated and poorly differentiated keratinocytes, and in order to assess the proliferative activity of all K10 and K6 subpopulations, simultaneous assessment of K6, K10, and DNA content is required. So far, a triple staining protocol had not been available. In the present study, we established a novel protocol for simultaneous measurement of K6, K10, and DNA content, which enables the characterization of the proliferative activity of several cellular subpopulations in epidermis. From 16 patients with psoriasis and from 15 healthy volunteers, punch biopsies were obtained. After preparation of single cell suspensions, cells were stained with the anti-keratin 10 IgG(1)-isotype monoclonal antibody RKSE60, with the anti-keratin 6 IgG(2a)-isotype monoclonal antibody LHK6B, and with the DNA fluorochrome TO-PRO-3 iodide. Isotype specific secondary antibodies conjugated with phycoerythrein (PE) and fluorescein isothiocyanate (FITC) were used as the second step in the staining procedure. Controls were measured omitting the primary antibodies, and gates were set in order to differentiate between the K10 and K6 subpopulations. Samples from both psoriatic patients and healthy volunteers were than measured. Owing to the IgG specificity of RKSE60 and LHK6B, no cross-reactivity was observed between these antibodies. The triple staining with RKSE60, LHK6B, and TO-PRO-3 iodide showed subpopulations of K10 expressing cells, K10/K6 co-expressing cells, and K6 only expressing cells. There was a significant difference in the proportion of K6 expression and K10/K6 co-expression between psoriatic and normal skin. Moreover, the proliferative activity of these subpopulations could be quantified by this protocol. We concluded that a triple staining protocol for the assessment of K6, K10, and DNA content, using the monoclonal antibodies LHK6B, RKSE60, and TO-PRO-3 iodide, supplies reliable and reproducible data for cellular studies on these keratins and for studying the proliferative activity of the subpopulations of these keratins in epidermis. Moreover, the present study showed that with respect to the proportion of K6, significant differences are present between psoriatic and healthy human skin.

Apoptosis has been indicated as a mechanism of T cell depletion in HIV-infected subjects and useful in monitoring disease progression. We investigated for the presence of apoptotic T lymphocytes in 130 HIV subjects in various stages of disease by the newly developed cell permeant DNA dye Apostain. Blood was collected in EDTA, lysed in buffered ammonium chloride, fixed in freshly prepared 1% paraformaldehyde and stored in aliquots at -80 degrees C. Samples were thawed and double stained with FITC conjugated-CD3 monoclonal antibody and Apostain. Flow cytometry was then performed and T cells gated on a CD3 versus side scatter dot plot. Normal samples treated in the same manner served to establish the boundary separating non-apoptotic from apoptotic cells. There was no statistically significant association between the proportion of subjects with detectable apoptotic cells and CDC clinical categories A, B and C at the time of admission to the study, although a trend toward a lower apoptotic rate in category A (A= 29%, B=40% and C=41%) was noticed. Conversely, CDC T cell categories 2 and 3 contained significantly higher proportions of Apostain positive patients (1=6%, 2=32% and 3=49%, P=0.072, by chi(2) test). Most importantly, Apostain test identified subjects at risk of disease progression during a 3.5-7 months follow-up in CDC category B and 2 (P=0.008 and P=0.0003, by Fisher's exact test, respectively). A similar, albeit not statistically significant trend was observed also in the other categories. Not requiring extensive manipulation of fresh samples nor cumbersome culture techniques, Apostain test appears suitable for identifying HIV subjects at higher risk of disease progression in clinical settings.

Background: The major diagnostic role of peripheral lymphocyte subset typing is to distinguish between malignant and reactive conditions.

Methods: The present study evaluates the screening efficacy of flow cytometric lymphocyte subset typing for the presence of a lymphoid malignancy. Four hundred samples were analyzed with a combination of anti-T-, B-, and natural killer (NK)-cell monoclonal antibodies.

Results: Two hundred and twenty (55%) samples showed a normal distribution of lymphocyte subsets, 73 (18%) samples exhibited unspecific alterations of lymphocyte subsets, 19 (5%) samples exhibited a reactive phenotype typical of Epstein-Barr virus/cytomegalovirus (EBV/CMV) infection, and 88 (22%) samples expressed a phenotype suggestive of lymphoma. The most predictive independent factor of a lymphoma-specific phenotype was the absolute lymphocyte count (P = 0.0001, odds ratio 73.225). Seventy-eight percent of samples containing >/=4 x 10(9)/l lymphocytes and 2% of samples with lymphocyte counts <4 x 10(9)/l exhibited a lymphoma-specific phenotype. The specificity of the referring clinical comment was the second best predictor of a lymphoma-specific typing outcome (P = 0.0001, odds ratio 19.589). The independent predictive values of lymphocyte morphology and of relative lymphocyte counts were of borderline significance.

Conclusions: The use of flow cytometric lymphocyte subset typing as a diagnostic screening method for lymphoma should be restricted to cases of unexplained elevation of absolute lymphocyte counts with or without morphological atypias and to cases with definite clinical symptoms of lymphoma.

Infection is very common in thalassemia and is one of the major causes of death. To date, it is not quite clear why these patients are susceptible to infection. In this study, lymphocyte immunophenotyping for CD3(+) (T-cells), CD3(+)CD4(+) (T-helper/inducer cells), CD3(+)CD8(+) (T-suppressor/cytotoxic cells), CD3(-)CD19(+) (B-cells), and CD3(-)CD16/56(+) (natural killer cells) subsets and expression of the activation antigen CD69 on CD3(+)CD4(+) and CD3(+)CD8(+) T-cells were determined in the whole blood of thalassemia patients, using a three-color flow cytometric technique. Results showed that only splenectomized beta-thalassemia/hemoglobin (Hb) E patients displayed a marked increase in absolute number of all lymphocytes. In addition, splenectomized beta-thalassemia/Hb E showed a significantly lower percentage of CD3(+) cells, with a corresponding increase in CD19(+) cells. These differences, when compared with normal subjects and other thalassemia patients, may be attributed to splenectomy. alpha-thalassemia patients, on the other hand, showed no significant difference from the normal group. While lymphocyte subsets in splenectomized beta-thalassemia/Hb E patients showed an abnormal distribution, T-cell activation in these patients was not different from the activation seen in normal subjects. This implies that thalassemia patients, during the steady state of disease, appear to have normal T-lymphocyte function with only moderate abnormalities of T- and B-lymphocyte subsets.

Background: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively.

Methods: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance.

Results: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug.

Conclusions: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

Background: Induction of apoptosis in adherent cell lines is associated with cell loss from the substratum. In this study the adenocarcinoma cell line, HT29, treated with indomethacin (400microM) has been employed as a model system to demonstrate how flow cytometric analysis can be used to quantify the changes that occur during this process.

Methods: Adherent and floating cell populations have been analyzed independently for effects on cell number, cell cycle characteristics and apoptosis using TUNEL assay and Annexin V binding. In addition apoptosis has been assessed using DNA laddering and morphology.

Results: Apoptosis was detected in adherent cells treated with indomethacin using Annexin V binding but not by other techniques employed in this study. In contrast, analysis of "floating" cells revealed the presence of apoptotic cells both in control and indomethacin treated cells using all the techniques employed. However quantification by flow cytometry showed that a significantly higher proportion of control "floaters" were late apoptotic/necrotic rather than apoptotic.

Discussion: The data here illustrate the need to interpret measures of apoptosis in adherent cell lines with care and the value of using flow cytometric techniques in the quantitative evaluation of the process.

Background: Multiparameter DNA flow cytometry using a one-laser bench-top flow cytometer has been restricted to three different colors. The two laser FACSCalibur has recently been introduced, allowing four-color analysis. Therefore, we optimized and extended our three-color method (Corver et al., 1994, Corver et al. 1996) to a four-color analysis of phenotypic intra-tumor heterogeneity using a bench-top flow cytometer.

Methods: First, the effect of a range of different propidium iodide (PI) and TO-PRO-3 iodide (TP3) concentrations on the coefficient of variation (CV) of the DNA histograms was measured using paraformaldehyde-fixed lysolecithin-permeabilized peripheral blood lymphocytes (PBLs) and SiHa and HeLa cervical cancer cells. Second, labeling freshly isolated cervical cancers from solid tumors was optimized with a mixture of anti-keratin antibodies. Third, the FACSCalibur hardware was modified, thereby allowing the simultaneous measurement of allophycocyanin (APC) fluorescence (FL4) in combination with FL3 pulse processing (FL3-W vs. FL3-A). The optimized procedure was then applied to cell suspensions from four different human cervical cancers to study phenotypic intratumor heterogeneity. Cell suspensions were simultaneously stained for DNA (PI, fluorescence) and three cellular antigens: (a) the epithelial cell-adhesion molecule (Ep-CAM; APC fluorescence), (b) keratin (R-phycoerythrin [RPE] fluorescence) to identify the epithelial fraction, and (c) vimentin (fluorescein-isothiocyanate [FITC] fluorescence) to label stromal cells.

Results: Overall, PI produced better CVs than did TP3. The optimal concentration of PI was 50-100 microM for all cells tested. Average CVs were 1.76% (PBL), 3.16% (HeLa), and 2.50% (SiHa). Optimal TP3 concentrations were 0.25-2.0 microM. Average CVs were 2. 58% (PBL), 5.16% (HeLa), and 3.96% (SiHa). Inter- or intra-DNA stem line heterogeneity of Ep-CAM expression was observed in the keratin-positive fractions. Vimentin-positive, keratin-negative cells were restricted to the DNA diploid fraction.

Conclusions: PI is a superior DNA stain to TP3 when using intact normal PBL and human cancer cells. Four-color high-resolution multiparameter DNA flow cytometry allows the identification of intratumor subpopulations using PI as DNA stain and FITC, RPE, and APC as reporter molecules. The FACSCalibur bench-top flow cytometer can be used for this purpose, allowing the application of this technique in clinical laboratories.

Background: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes.

Methods: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results.

Results: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis.

Conclusions: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.

Background: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres.

Methods: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype.

Results: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases.

Conclusions: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.

Background: An essential parameter that describes the quality of liposome suspensions is the mean size, respectively the size distribution. Currently several analytical methods including laser light scattering techniques (LLST) are being employed.

Methods: Here we present an alternative technique using flow cytometry (FCM) to characterize uni- and polydisperse suspensions. As model liposomes preparations containing dipalmitoylphosphatidylcholine (DPPC) were used. A constant number of particles (1,500/s) in the fluid stream and a representative number of 10,000 particles of each sample was measured. Fluorescence-labeled latex beads were measured identically, and their side scatter signals were calibrated and correlated to the results obtained with liposome vesicles.

Results: Evaluation of the measurement and validation of the FCM results in comparison to LLST confirm the reliability of results obtained with our method. Latex beads in the range of 100-1000 nm were used for calibration to classify liposomes. Although measurement characteristics and calculation in both methods are basically different, very good agreement of the results was achieved.

Conclusions: Demonstration of stability, reproducibility, and reliability of results make the employment of this method acceptable for an adequate routine analysis technique.