Characterization of human monocyte subpopulations by flow cytometry.

Human monocytes separated from peripheral blood by Ficoll-Hypaque and by adherence to serum-coated dishes show a bimodal volume distribution measured with a fluorescence-activated cell sorter. In the first peak of size distribution histogram of living mononuclear cells, lymphocytes and small monocytes were characterized by latex phagocytosis and non-specific esterase staining, whereas in the second peak the large monocytes dominated. The percentage of esterase stained small monocytes was lower than that of the large ones. Parallel to these data, the rate of the FDA hydrolysis of the small monocytes was lower than that of the large ones. The majority of the large monocytes reacted with sensitized sheep red blood cells (sSRBC) while only the minority of the small monocytes bound sSRBC. Scatchard plots on the binding of fluorescein isothiocyanate (FITC)-labelled human monoclonal IgG1 to the two subpopulations indicated similar association constants. K = 1 . 2 +/- 0 . 3 X 10(5) M-1. The number of Fc receptors was significantly different for the small (3 . 3 +/- 0 . 6 X 10(5)) and the large monocytes (10 +/- 1 X 10(5)).     

Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis

Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogeneous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2Kk) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and gamma 2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages.     

Characterization of rickettsial attachment to host cells by flow cytometry.

The mechanisms of rickettsial attachment have been studied by measuring quantitative changes in rickettsial binding to host cells by flow cytometry after different treatments of the rickettsiae and host cells. Time-dependent binding of Rickettsia conorii to host cells was demonstrated by the increasing intensity of host cell surface fluorescence of rickettsia-host cell combinations when examined with a rickettsia-specific monoclonal antibody. More than 70% of host cells had intensity of fluorescence above the threshold value after 10 min of incubation, owing to rickettsiae bound to the cell surface, and the greatest fluorescence intensity indicative of binding occurred at 20 min. The binding kinetics was rickettsial dose dependent. The binding of rickettsiae to host cells was greatly decreased when host cells or rickettsiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 5 or 15 min, respectively. Rickettsiae that were heated at 56 degrees C for 15 min lost more than 80% of their ability to attach to host cells. R. rickettsii, an organism closely related to R. conorii, competitively inhibited the attachment of R. conorii (51% inhibition when mixed in equal numbers). These results indicate that the rickettsial binding structures are trypsin and heat sensitive and likely to be surface proteins.     

Characterization and use of a rabbit-anti-mouse VPAC1 antibody by flow cytometry

Vasoactive intestinal peptide receptor-1 signaling in lymphocytes has been shown to regulate chemotaxis, proliferation, apoptosis and differentiation. During T cell activation, VPAC1 mRNA is downregulated, but the effect on its protein levels is less clear. A small number of studies have reported measurement of human VPAC1 by flow cytometry, but murine VPAC1 reagents are unavailable. Therefore, we set out to generate a reliable and highly specific α-mouse VPAC1 polyclonal antibody for use with flow cytometry. After successfully generating a rabbit α-VPAC1 polyclonal antibody (α-mVPAC1 pAb), we characterized its cross-reactivity and showed that it does not recognize other family receptors (mouse VPAC2 and PAC1, and human VPAC1, VPAC2 and PAC1) by flow cytometry. Partial purification of the rabbit α-VPAC1 sera increased the specific-activity of the α-mVPAC1 pAb by 20-fold, and immunofluorescence microscopy (IF) confirmed a plasma membrane subcellular localization for mouse VPAC1 protein. To test the usefulness of this specific α-mVPAC1 pAb, we showed that primary, resting mouse T cells express detectable levels of VPAC1 protein, with little detectable signal from activated T cells, or CD19 B cells. These data support our previously published data showing a downregulation of VPAC1 mRNA during T cell activation. Collectively, we have established a well-characterized, and highly species specific α-mVPAC1 pAb for VPAC1 surface measurement by IF and flow cytometry.     

Detection of platelet alloantibodies by flow cytometry. Characterization and clinical significance

A flow cytometric technic was developed to detect platelet alloantibodies in patients who had received multiple platelet transfusions. All clinically alloimmunized patients had IgG alloantibodies, whereas the nonalloimmunized patients were within the range of the normal controls. There was a negative correlation between the IgG level and the platelet increment. IgM and IgA alloantibodies also were detected in some patients. When they were present in addition to IgG, the platelet increment appeared to be reduced further.     

An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry

A comparison was made between a manual and automated method for preparing single cell suspensions from various types of human biopsy samples. The automated method uses the shearing action of fluid movement generated by a reciprocating paddle system. When compared on 11 samples, the automated instrument always isolated cells in less time, usually requiring only 15-30 seconds in contrast to 10-15 minutes for the manual method. The time comparisons involved "set up" time of the required minor equipment as well as the time to make a complete single cell suspension from the portion of the biopsy sample sent to the Flow Cytometry Lab so extra cells could be used for other purposes and cryogenically stored for future reference. Cells isolated by the automated method from various B-lymphocytic and T-lymphocytic malignancies were still viable and could be successfully immunophenotyped by flow cytometry. The immunophenotyping results were compared for both cell isolation methods on seven of these samples, and the results were comparable. The automated technique has now been used to satisfactorily immunophenotype more than 50 biopsy samples. The automated method should represent a significant aid for clinical flow cytometry laboratories performing immunophenotyping tests by efficiently preparing single cell suspensions in a few seconds instead of minutes. Furthermore, the automated method uses a closed sterile bag system that helps minimize the exposure of personnel to potential infectious material present in biopsy samples and prevents external contamination of cell suspensions. The automated technique proven successful for immunophenotyping may also be helpful for related procedures involving hematopoietic malignancies such as DNA content analysis and cell functional assays by flow cytometry, as well as other assays such as tissue typing, gene probe, and in vitro chemosensitivity assays.     

Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67

A novel procedure for determining the growth fraction of cell suspensions by flow cytometry is described. This method identifies proliferating cells by binding the monoclonal antibody Ki-67 to a nuclear antigen present in all cells that are in the G1, S, G2, and M phase of the cell cycle, but not in the G0 phase. In a kinetic study of Na cell line U937 using concanavalin A for stimulation of peripheral blood mononuclear cells, a steady increase of Ki-67 positive cells evaluated by flow cytometry was observed. Simultaneously, the [3H]thymidine uptake of the ConA blasts was measured and compared to the expression of Ki-67. A linear correlation between the percentage of Ki-67 positive cells and the log transformed counts per minute was demonstrated, and staining with Ki-67 detected cell proliferation with the same sensitivity as 3H-TdR uptake. In addition, it was possible to stain Ki-67-labelled cells with a second cell marker if a second fluorescent dye coupled to an antibody was used. This provided the opportunity to define precisely the phenotype of proliferating cells. Conversely, the number of proliferating cells expressing certain preselected surface markers could be easily determined.     

Characterization of the TSU-PR1 cell line by chromosome painting and flow cytometry

TSU-PR1 was originally reported as a prostatic carcinoma cell line derived from a lymph node metastasis. Recently, however, this cell line was reported to be derived from T24 bladder carcinoma cells, and thus further definition of its origin is needed. Conventional cytogenetic study of TSU-PR1 showed aneuploidy, ranging from 65 to 86 chromosome with a modal number of 80, and with 10 marker chromosomes, thus conventional cytogenetics cannot be used to determine which chromosomes or regions of chromosomes are critical in cancer development and progression of this cell line. The present study was conducted to characterize genetic changes of the cell line using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and flow cytometry. CGH results showed that green-to-red fluorescence ratios were within the range of 0.85-1.15, except for a few chromosomes, which reflected near tetraploidy in TSU-PR1. Flow cytometric analysis of TSU-PR1 revealed a DNA index of 3.46n, which is close to the 3.48n calculated from a modal number of 80. The copy numbers of chromosomes 4, 6, 7, 17, and 20 determined by the DNA index and the CGH analyses were 2.85 +/- 0.09, 3.22 +/- 0.77, 3.01 +/- 0.26, 4.05 +/- 0.44, and 4.99 +/- 0.48, respectively. These numbers are also in accordance with the chromosome copy numbers determined with FISH: 2.98 +/- 0.23, 2.91 +/- 0.44, 2.74 +/- 0.44, 3.93 +/- 0.38, and 5.05 +/- 0.78 for chromosomes 4, 6, 7, 17, and 20, respectively (P > 0.05).     

Analysis of megakaryocytes by flow cytometry

A flow cytofluorometric measurement of megakaryocyte ploidy has been adapted from Tomer's method. Briefly, bone marrow is aspirated through a medium containing theophyllin, prostaglandin-E1 and other antiaggregant agents. Megakaryocytes-enriched buffy coats are recovered. Megakaryocytes are then stained for GP IIIa coupled with FITC and for DNA (propidium iodide). A Becton Dickinson FACScan flow cytometer is used for measuring the ploidy distribution of GP IIIa positive cells. The method we developed presents several advantages. Firstly, the time required is greatly decreased in comparison with other studies. Secondly, the washing steps are limited in number allowing a diminution of the cell loss. Thirdly, the method ensures a better megakaryocyte preservation. Finally, selection of megakaryocytes by ploidy and expression of GP IIIa can be made easily because of the simultaneity of the two stainings as well as by the use of a precise gating on the flow cytometer. Based on these results, we conclude that the present method provides a better means for the isolation and analysis of human normal megakaryocytes. This technique has been applied to the analysis of megakaryocyte populations from patients with abnormal platelet counts. In chronic myeloid leukemia patients, analysis of ploidy distribution shows a shift toward the low ploidy while in patients with immune thrombocytopenic purpura, polycythemia vera and essential thrombocythemia the ploidy distributions are shifted toward the high ploidy.     

Measurement of alloantibody by flow cytometry

A new method based on flow cytometry has been developed to determine IgG alloantibodies in the serum of immunized rodents. The method utilizes target lymphoid cells, diluted serum and labeled anti-mouse or anti-rat IgG antibodies. In serum from highly immunized animals alloantibodies could be demonstrated up to a dilution of (3 x 10(4))-1 which makes the test approximately 300 times more sensitive than a simultaneously performed complement-dependent cytotoxicity assay (CDCA). Moreover a linear relationship between the amount of alloantibody and the log value of the mean fluorescence of the target cells was found. This linearity permits the comparison of alloantibody production between individual samples by comparing the mean fluorescence values. The reproducibility of the assay was excellent since the coefficients of variation for all dilutions were less than 15%. By using two-color fluorescence the method can discriminate alloantibodies directed against class I and class II MHC antigens. Finally, in addition to its high sensitivity and good reproducibility, the method was found to be at least twice as fast as CDCA.     

Phagocytosis: measurement by flow cytometry

Defects in phagocyte function or in the interactions between phagocytes, microorganisms and serum factors are associated with increased susceptibility to infection. Flow cytometry (FCM) offers rapid and reproducible measurements of single cells in suspension and, following staining with one or more fluorochromes, simultaneous biochemical and functional examinations of the complex process of phagocytosis. FCM techniques have been used for more than two decades to evaluate phagocyte cellular defects, as well as species-specific serum opsonic activities during disease and after vaccination. Recently, multiparameter assays have been developed to reveal the antigen-specificity of opsonophagocytic responses. This review presents basic methodological principles of FCM quantitation of phagocytosis and intracellular oxidative burst, and assays to evaluate species-specific and antigen-specific opsonophagocytosis. The calculations performed to present opsonophagocytosis results, as well as technical and methodological challenges are discussed, and examples of applications are presented.     

Fluorescence polarization assay by flow cytometry

Fluorescence polarization measurement on cell suspensions provides a highly sensitive means for detecting subtle changes in the cells, such as occur early after lymphocyte activation or on malignant transformation. We review here the principles of fluorescence polarization, its measurement by a commercially available flow cytometer and application of such assays especially in cellular immunology.     

Red cell age by flow cytometry

A method is presented for the use of red cell markers to assess the age of red cells in clinical samples. The reticulocyte count and its variants are already in clinical use to measure the number of young circulating red cells, but tools have not been put into place for studying the overall distribution of red cell age. These data could be of significant value, not merely for hematologic investigations, but as a part of infectious disease, renal, and toxicologic studies.     

Characterization of bladder tumours by multiparameter flow cytometry with special reference to grade II tumours

Sixty-three human transitional cell carcinomas of the urinary bladder were studied by multiparameter flow cytometry (FCM). The cellular DNA content, the cellular protein content, the fraction of cells in S phase, and the nuclear size were registered and correlated to histological grade (WHO) and histologically determined infiltration through the basement membrane. Aneuploidy was found in the great majority of grade III tumours, but in only 24% of grade II tumours. A new, combined variable, viz. the cellular DNA to protein ratio, indicated a possibility for further subdivision of the tumours. Grade II tumours, which constitute a rather heterogeneous group with regard to prognosis, could be classified in two subgroups: One group of diploid tumours with the FCM characteristics of grade I tumours, and another group of diploid and aneuploid tumours with the characteristics of grade III tumours. Infiltration was most frequently seen in the latter subgroup. The putative prognostic relevance of such a subdivision will be the subject of a future study. Compared to FCM measurement of DNA alone, multiparameter FCM, including measurement of the total cellular protein content, has given additional information that may be of prognostic value.     

Immunophenotyping of cytologic specimens by flow cytometry

Immunophenotyping by flow cytometry is well established as an ancillary technique in the diagnosis of hematopoietic neoplasms. However, flow cytometry is rarely performed on cytologic specimens because most cytologists are more comfortable with direct microscopy and believe that there is inadequate cellularity for analysis. Paradoxically, cytologic material is usually cell suspensions making it ideal for flow cytometry. In order to evaluate the usefulness of immunophenotyping cytologic specimens by flow cytometry, we retrospectively reviewed all cytologic specimens submitted to our flow cytometry unit from 1988 to 1991. Thirty-one cerebrospinal fluid specimens were analyzed. There were inadequate cells for analysis in 15 cases. Five showed a monoclonal proliferation; 11 were nondiagnostic. A range (r) of one to six cell surface markers were performed. Thirty-two body cavity fluids were analyzed: 7 peritoneal, 19 pleural, 2 pericardial, and 4 bronchoalveolar lavage. There were cells to analyze in all cases. Seven had a monoclonal proliferation; 25 were nondiagnostic (r = 4-21 markers performed). One hundred eighteen fine needle aspirates (FNA) were reviewed; 58 FNA were radiologically guided, 60 were superficial lesions. There were inadequate cells for analysis in two cases. Sixty-one demonstrated a monoclonal proliferation; 55 were nondiagnostic (r = 1-22 markers performed). We conclude that immunophenotyping by flow cytometry is of limited value for cerebrospinal fluid analysis and that knowledge of previous immunophenotyping studies is essential for correct analysis; analysis of body cavity fluids is easily performed but less often demonstrates a monoclonal proliferation. Immunophenotyping by flow cytometry is a valuable adjunctive technique for FNA and yields adequate cells for analysis.     

Detection of intracytoplasmic antigens by flow cytometry

A new technique is described for the detection of intracellular antigens by immunofluorescence and flow cytometry. The technique utilizes lysolecithin (lysophosphatidylcholine), a naturally occuring phospholipid, to permeabilize cell membranes and allow antibodies to reach intracellular antigens. The technique is rapid and sensitive, and retains sufficient integrity of the cells being treated to enable differentiation of cell types on the basis of light scatter (e.g., lymphocytes from monocytes). Permeabilization of cells following lysolecithin was assessed using standard techniques including trypan blue exclusion, propidium iodide staining, and hydrolysis of fluorescein diacetate. Lysolecithin treatment was accompanied by only minimal increases in non-specific background fluorescence, and no increase in autofluorescence. Our studies have demonstrated that lysolecithin treatment of human mononuclear cell populations permits flow cytometric analysis of cytoskeletal structures, including intermediate filaments, as well as cytoplasmic immunoglobulin. Studies currently in progress in our laboratory have demonstrated broad intracellular reactivity with some monoclonal antibodies identifying leukocyte differentiation and tumor-associated antigens. These findings contrast with the more restricted expression of these antigens on the cell surface and demonstrate not only the value of the lysolecithin technique but also the importance of the study of intracellular antigens in our overall understanding of the specificity, distribution, synthesis, and function of cellular antigens.     

Quantitation of baculovirus particles by flow cytometry

A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.