Selective trapping of live and dead mammalian cells using insulator-based dielectrophoresis within open-top microstructures

The manipulation of biological cells is essential to many biomedical applications. Insulator-based dielectrophoresis (iDEP) trapping consists of insulating structures which squeeze the electric field in a conductive solution to create a non-uniform electric field. The iDEP trapping microchip with the open-top microstructures was designed and fabricated in this work. For retaining the merit of microfabrication, the microelectrodes were deposited on the substrate to reduce the voltage required, due to the shortened spacing between them. The dielectrophoretic responses of both live and dead HeLa cells under different frequencies (100 Hz, 1 kHz and 1 MHz) have been investigated herein. The live cells exhibited negative dielectrophoresis at low frequencies of 100 Hz and 1 kHz, but a positive dielectrophoretic response with the frequency at 1 MHz. As for dead cells, positive dielectrophoretic responses were shown at all the frequencies applied. Therefore, selective trapping of dead HeLa cells from live cells was achieved experimentally at the frequency of 1 kHz. The open-top microstructures are suitable for trapping cells or biological samples, and easily proceeding to further treatment for cells, such as culturing or contact detection. The intensity of the emitted light during fluorescent detection will not suffer interference by a cover, as it does not exist herein.     

Flow cytometric analysis of pulmonary fluids and cells for the detection of malignancies.

Cells from a pulmonary or bronchial origin were analyzed with flow cytometry to assess the sensitivity and specificity of this method in diagnosing malignancy. In all instances, cells submitted for flow cytometry analysis were excess cells from specimens submitted for routine cytology. Less than 3% of all samples were rejected for insufficient material. Overall sensitivity from all sources was 86%, specificity 96%. Although cytology results were the standard for determining accuracy of flow cytometry, in a few patients cytology appeared normal and initial evidence for malignancy was obtained from flow cytometry. For this reason, flow cytometry may be a valuable adjunctive technology in the diagnosis of malignancy.     

Flow cytometric analysis using SYBR Green I for genome size estimation in coffee

Plant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G₀/G₁ peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57-2.85%) compared to those stained with propidium iodide (2.75-4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide.     

Identification of live cells for flow cytometric analysis of lymphoid subset proliferation in low viability populations

Combined analysis of single cell DNA content and immunofluorescence by flow cytometry is complementary to tritiated thymidine analysis of cellular proliferation, allowing detailed dissection of particular cell types in a mixed population which respond proliferatively to selective stimuli. However, in vitro culture of primary immune cells (e.g., mouse spleen or lymph node) for periods of 24-72 h frequently results in a considerable fraction of non-viable cells which bind antibodies non-specifically, resulting in altered immunofluorescence distributions, inaccurate distinctions between positive and negative cells, and sometimes in misleading DNA distributions. Forward angle light scatter cannot readily be used to distinguish live from dead cells in this case because of the heterogeneous size distributions characteristic of cultured populations. We describe a method which uses treatment with DNAase prior to immunofluorescence staining to allow more accurate distinction between live and dead cells. This treatment markedly reduces the intensity of DNA staining for non-viable cells, providing complete live/dead discrimination and improved ability to analyze the proliferative status of specific cell subtypes in low viability cultures.     

Flow cytometric analysis of virus-infected cells and its potential use for screening antiviral agents.

Virus-infected cells were analyzed using multiparameter flow cytometry. Two virus-cell systems were investigated: HSV-1-infected VF cells and influenza C virus JHB/1/66-infected MDCK cells. Analysis included the measurement of the appearance of virus specific antigens. On individual cells, with polyclonal antibodies, antigens were first detected at 12 h p.i., and the numbers of labeled cells were followed up to 96 h p.i. The efficacy of four antiviral agents was tested with this system. The results were in good agreement with those of plaque reduction tests and indicated that this new method may be extremely useful for the correlation of viral and cellular events with antiviral activity. Finally, it was demonstrated that infected cells in both systems have a considerably greater volume than non-infected cells.     

Flow cytometric analysis of T cell proliferation in a mixed lymphocyte reaction with dendritic cells

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells. They can be generated in vitro from CD14+ cells, and also from CD34+ progenitor cells. Although T cell proliferation using [3H] thymidine incorporation assay has been used widely to check DC function, this technique only provides limited information about the T cell proliferation. Here, we describe a novel method for quantitative analysis of T cell proliferation using flow cytometry. MATERIALS AND METHODS: DCs were generated from CD14+ cells from six healthy blood donors. Monocytes were isolated using positive selection with magnetic cell sorting (MACS) and then cultured with IL-4, GM-CSF, IL-1beta, IL-6, TNF-alpha and PGE(2) to yield fully mature DCs. Allogeneic naive T lymphocytes with known mismatches in HLA classes I and II were cocultured with DCs. Naive T cells without DC stimulation served as negative controls. T cells were harvested on days 0, 3, 5, 7, 9, 11 and analysed by flow cytometry. CD3-ECD and CD4-fluorescein isothiocyanate (FITC) or CD8-FITC antibodies were used to distinguish T cell subsets, whereas T cell activation was measured by assessment of HLA-DR, CD45RO, CD25 and CD71 expression. For T cell quantification, fluorescent microparticles were used. Dead cells were excluded with 7-AAD. The bromdeoxyridine (BrdU)-incorporation ELISA procedure was also performed in order to compare with the T cell proliferation assay with regard to absolute cell counts and CD71 expression. RESULTS: The initial T cell concentration on day 1 was 203.9+/-39.7 (173-265) CD3+/CD4+ cells/micro l and 184.5+/-41.6 (148-260) CD3+/CD8+ cells/micro l. The maximal T cell proliferation was recorded on day 7 with a five- to tenfold T cell expansion which resulted in 1994.9+/-383 (1446-2404) CD3+/CD4+ cells/micro l and 944+/-303.7 (560-1483) CD3+/CD8+ cells/micro l. Furthermore, activation markers of both cell lineages were upregulated and reached maxima on days 7 (CD71) and 9 (CD25, HLA-DR). T cell count/micro l as well as CD71 expression both correlated significantly with BrdU incorporation. CONCLUSION: Flow cytometric analysis permits simple, precise and rapid quantification of T cell proliferation in a mixed lymphocyte reaction with DCs. Activation, proliferation and cell viability can be simultaneously determined. CD71 is particularly well suited as an activation marker for the simultaneous measurement of T cell proliferation. Thus, specific T cell subsets involved in antigen-specific proliferation can be evaluated in detail.     

Assays for mitotic chromosome condensation in live yeast and mammalian cells

The dynamic reorganization of chromatin into rigid and compact mitotic chromosomes is of fundamental importance for faithful chromosome segregation. Owing to the difficulty of investigating this process under physiological conditions, the exact morphological transitions and the molecular machinery driving chromosome condensation remain poorly defined. Here, we review how imaging-based methods can be used to quantitate chromosome condensation in vivo, focusing on yeast and animal tissue culture cells as widely used model systems. We discuss approaches how to address structural dynamics of condensing chromosomes and chromosome segments, as well as to probe for mechanical properties of mitotic chromosomes. Application of such methods to systematic perturbation studies will provide a means to reveal the molecular networks underlying the regulation of mitotic chromosome condensation.     

Flow cytometric quantitative evaluation of phagocytosis by human mononuclear and polymorphonuclear cells using fluorescent nanoparticles

The use of fluorescent polymethacrylic nanoparticles (0.3 micron) as a flow cytometric reagent in the quantitative evaluation of phagocytosis by human mononuclear and polymorphonuclear cells is described. The preparation of the nanoparticles, by emulsion copolymerization of methacrylic monomers, and their physicochemical properties are briefly summarized. Nanoparticles coupled with a fluorescent agent (ethidium bromide) were used in a flow cytometric assay to study opsonin-independent phagocytosis by human polymorphonuclear cells and by human monocytes. The phagocytosis of nanospheres by monocytes was determined by flow cytometry from the fluorescence distribution and ingestion was visualized by scanning and transmission electron microscopy. One possible application of the fluorescent nanoparticles is the simultaneous analysis of cell surface antigens and cell phagocytic activity.     

Flow cytometric analysis of gamma delta T cells and natural killer cells in HIV-1 infection.

We have previously shown that HIV-1 seropositivity is associated with an increase in the difference between the number of CD3+ lymphocytes and the total number of CD4+ and CD8+ lymphocytes [CD3 - (CD4 + CD8)] among peripheral blood lymphocytes (PBL). To investigate the cellular basis of this increase, PBL from seronegative (SN) and AIDS-free seropositive (SP) homosexual men and intravenous drug users were analyzed by two-color flow cytometry. Results showed that SP compared to SN manifested the expected elevation in calculated [CD3 - (CD4 + CD8)] cells (87 vs 28 cells/mm3; P less than 0.001). Only small differences in lymphocyte populations that could contribute to this increase were observed, namely lymphocytes expressing the CD3+CD4-CD8-phenotype (67 vs 56 cells/mm3; P greater than 0.10) or the CD8dim phenotype (135 vs 142 cells/mm3; P greater than 0.10). However, SP had significantly lower numbers of cells expressing the CD56+CD3- phenotype characteristic of natural killer cells (81 vs 170 cells/mm3; P less than 0.001) and significantly higher numbers of T cells expressing the gamma delta T cell receptor (TCR) (81 vs 62 cells/mm3; P = 0.10). The latter difference was primarily due to higher numbers of cells coexpressing gamma delta-TCR and low levels of CD8 (27 vs 15 cells/mm3; P = 0.009). These data suggest that HIV-1 seropositivity is associated with low numbers of natural killer cells and high numbers of CD8+ gamma delta-TCR lymphocytes. Changes in these populations may reflect altered host defense against HIV-1 or altered T cell kinetics in the presence of HIV-1 infection.     

CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers

Background

Diagnostic tests for allergy rely on detecting allergen-specific IgE. Component-resolved diagnostics incorporate multiple defined allergen components to improve the quality of diagnosis and patient care.

Objective

To develop a new approach for determining sensitization to specific allergen components that utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flow cytometry.

Methods

Recombinant forms of Lol p 1 and Lol p 5 proteins from ryegrass pollen (RGP) and Api m 1 from honeybee venom (BV) were produced, biotinylated, and tetramerized with streptavidin-fluorochrome conjugates. Blood samples from 50 RGP-allergic, 41 BV-allergic, and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation of basophil allergen binding and activation.

Results

Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves. Staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single-tube assay enabled rapid detection of sensitization to both Lol p 1 and Lol p 5 in RGP-allergic patients and discriminated between controls, BV-allergic, and RGP-allergic patients.

Conclusion

Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease, and the assay can be multiplexed for a component-resolved and differential diagnostic test for allergy.

     

Flow cytometric analysis of lymphocyte phenotypes in AIDS using monoclonal antibodies and simultaneous dual immunofluorescence

Simultaneous dual immunofluorescence and flow cytometry was used to study sixteen lymphocyte phenotypes in 209 men including: healthy homosexuals, lymphadenopathy patients (LAN), and AIDS patients. Significant differences between the distribution of lymphocytes in healthy homosexuals and healthy heterosexuals were decreased percentages of helper/inducer T cells (Leu 3), increased cytotoxic/suppressor T cells (Leu 2), and consequently a decreased Leu 3/Leu 2 ratio. The increased Leu 2 cells were identified as functionally cytotoxic subset Leu 2+ 15- phenotype rather than suppressor cells which are Leu 15+. Leu 2 and Leu 3 bearing cells exhibited an excess of membrane-bound immunoglobulins which were easily elutable at 37 degrees C. An increased percentage of an HLA-DR framework determinant bearing T cells were also detected. Within the NK cell family, Leu 7 cells were moderately increased and the functionally unidentified Leu 2+ 7+ population was strikingly elevated. LAN or AIDS patients were compared to healthy homosexual controls. Lower percentages of Leu 3 cells and higher percentages of Leu 2 cells were evident in LAN patients. These subsets were similar in LAN and AIDS patients. The increase in Leu 2+ cells was due to the Leu 2+ 15- cytotoxic subset. Fewer T cells had immunoglobulin in LAN and AIDS. A definite increase in Leu 2+ DR+ cells but not Leu 3+ DR+ cells occurred in AIDS compared to LAN or healthy controls. NK cell changes already present in healthy homosexuals persisted in LAN and AIDS patients. No differences in the distribution of B cells was detected in any intergroup comparisons. Changes in monocytes or pan-T cells were relatively insensitive measures of immunologic alterations among any of the groups. These results indicate many of the changes in lymphocyte subsets seen in AIDS and LAN subjects are already present in a carefully screened population of healthy homosexuals in San Francisco. Many of the changes in Leu 2 and NK family of cells suggest a possible adaptive response to viral or neoplastic challenge. Whether these interesting phenotypic alterations relate to functional changes in response to such challenge of the identified subsets waits further investigation.     

Flow cytometric DNA analysis of gastric cancer--correlation with histology and clinical outcome.

Nuclear DNA content was analysed by means of flow cytometric measurements in 103 patients with gastric carcinomas, using paraffin-embedded archival tissue. DNA aneuploidy was found in 40 cases (38.8%). The mean DNA index of aneuploid tumors was 1.45(range 1.13 to 2.37). No significant association between ploidy and either age, sex, tumor location, size, stage, growth pattern, or histologic type was found. However, the incidence of aneuploidy was higher in high grade carcinomas than in low grade carcinomas; the incidence of aneuploidy was 10%, 68.8%, and 45.8% for Grade II, III, and IV carcinomas, respectively, as compared with Grade I carcinomas which were all diploid. On statistical analysis, Abnormal cellular DNA content was significantly correlated with high histologic grade (P < 0.005). Patients with aneuploid cancer (39.2%) had a poorer prognosis than those with diploid cancer (70.0%) based on (P < 0.01). The 2-year survival rate for advanced gastric carcinoma. Therefore, DNA ploidy might be a useful prognostic factor in cases of advanced gastric cancer.     

Ovary and ovulation: Flow cytometric analysis of granulosa cells from follicular fluid after follicular stimulation

Granulosa cells are not easily accessible, unless they are examinedin follicular fluid after oocyte retrieval. These samples areusually contaminated with blood. We have set up a general techniquefor analysis of granulosa cells without physically separatingthem from blood cells. The sample is stained with CD45, whichis a pan-leukocyte marker, and granulosa cells are consecutivelyselected as CD45 negative during flow cytometric analysis. Analysisof forward scatter of the granulosa cells, which is correlatedto cell size, shows a wide size range throughout the whole populationrather than two distinct populations as previously suggested.     

Flow cytometric and functional analysis of mononuclear cells infiltrating the liver in experimental autoimmune hepatitis.

Experimental autoimmune hepatitis was produced by immunizing Wistar rats with syngeneic liver proteins. Mononuclear cells infiltrating the liver tissue were identified by immunohistochemical techniques using monoclonal antibodies specific for subpopulations of rat lymphocytes. The strong infiltration of CD8+ cytotoxic T lymphocytes (CTL) were found in the portal areas. Subpopulations of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes were identified by flow cytometry. Flow cytometric analysis revealed the presence of CD5- and CD8+ lymphocytes in the liver tissues. Mononuclear cells infiltrating the liver were isolated from Wistar rats having autoimmune hepatitis to determine whether those exhibit cytotoxicity against syngeneic hepatocytes; they exhibited cytotoxicity against isolated syngeneic hepatocytes, but failed to lyse K562 cells, syngeneic concanavalin A-activated splenocytes and allogeneic hepatocytes. Depletion of CD8+ T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating into the liver against syngeneic hepatocytes. These findings support the idea that liver cell injury in experimental autoimmune hepatitis may at least in part be mediated by CTL.     

Flow cytometric methods for the analysis of human basophil surface antigens and viability

Fluorescence and flow microfluorometric methods have been established for the detection and evaluation of IgE-bearing human leukocytes in various cell preparations including those where basophils are present at low percentages. Quantitative techniques for the determination of basophil purity, viability, and cell surface antigens including IgE are described. Use of these methods will facilitate the identification and phenotypic analysis of human IgE-bearing cells in a wide variety of biological fluids.     

Flow cytometric analysis of surface light chain expression patterns in B-cell lymphomas using monoclonal and polyclonal antibodies

Light chain (LC) expression by flow cytometry (FC) in B cell non-Hodgkin lymphomas (B-NHLs) can occasionally be detected with one anti-LC antibody but not with another. We retrospectively analyzed 564 four-color FC files from B-NHLs, assessing LC staining with monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs). Discrepancies in LC expression between mAbs and pAbs were present in 9.2% of cases, mainly in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; 11.1%), diffuse large B-cell lymphoma (DLBCL; 10.2%), follicular lymphoma (9.5%), and mantle cell lymphoma (11.1%) and most frequently in body fluids. Equal proportions of cases were LC+ only with pAbs (4.8%) or mAbs (4.4%). Negative LC expression with both antibodies was present in 7.5% of cases, most frequently in DLBCL (21.6%) and body fluids (27.6%). Evaluation with both mAbs and pAbs increases the sensitivity for LC detection, with no single reagent outperforming the other, although CLL/SLL preferentially showed LC expression with pAbs.     

肺癌细胞DNA含量与肺癌病理形态学及预后的关系

DNA content of paraffin-embedded tumour material from 110 patients with lung carcinoma was measured by means of flow cytometry. Aneuploidy was found in 78% of the patients. The mean DNA ploid, the percentage of hyperdiploidy and the percentage of multiploidy in small cell carcinoma and large cell carcinoma were higher than those obtained in squamous cell carcinoma and adenocarcinoma. These values were also higher in grade III squamous cell carcinoma and adenocarcinoma as compared with grade I and II squamous cell carcinoma, and adenocarcinoma. They were also higher in lung carcinoma with the primary tumor size greater than or equal to 3 cm than that less than 3 cm. The five-year survival rate and mid mean survival time of patients with hyperdiploidy were significantly lower than those of patients with diploidy and hypodiploidy. Conclusively, DNA content is considered closely related to the degree of malignancy of lung carcinoma and may be adapted as a reliable criterion in estimating the prognosis of lung carcinoma.     

Flow cytometric DNA-quantification of three-color immunophenotyped cells for subpopulation specific determination of aneuploidy and proliferation

A method is described for three-color immunophenotyping and simultaneous DNA-quantification using a flow cytometer equipped with a 488-nm argon laser and a mercury lamp (UV). The approach includes reproducible immunophenotyping comparing antigen expression before and after cell manipulation for DNA-measurement. The coefficients of variation after DNA-staining (CV=3.13 for T-cells in peripheral blood and CV=3.38 for T-cells in bone marrow) were adequate for exact DNA-analysis. For aneuploidy detection, a true internal standard was established measuring, for example, the DNA-content of T-cells in B-cell disease simultaneously with the DNA-content of the malignant cells. Using this method, aneuploidies could be unequivocally detected in 17 out of 24 patients with multiple myeloma. Furthermore, intratumor heterogeneities in DNA-content and antigen expression could be recognized, allowing an exact separation of tumor cells and normal hematopoiesis. The study also demonstrated the importance of exact immunophenotypic characterization of lymphocyte subpopulations and the determination of their specific proliferation, for example after proliferation induction in cell cultures. Future studies should address the applicability of this rather simple multiparameter approach for simultaneous immunophenotyping and DNA-measurement especially in the detection of minimal amounts of aneuploid cells after chemotherapy.     

Flow cytometric analysis of DNA ploidy and S-phase fraction in breast cancer using cells obtained by ex vivo fine-needle aspiration: an optimal method for sample collection

A total of 203 primary invasive breast cancers were sampled by ex vivo fine-needle aspiration (FNA), directly yielding adequate single cell suspensions for flow cytometric DNA analysis in 194 (96%). Labor-intensive and time-consuming steps of mechanical and enzymatic cellular disaggregation required by the use of fresh, frozen, or paraffin-embedded tissue were avoided, thereby minimizing preparation time. Conservation of tumor tissue allowed for the sampling of very small breast cancers. DNA ploidy and S-phase fraction data were comparable to flow cytometric data reported in other breast cancer studies using various sampling methods. Ex vivo FNA is the easiest and fastest method for sampling breast cancers for flow cytometric DNA analysis.