Flow Cytofluorimetry Research Articles

Lymphocyte subclasses in pregnancy induced hypertension.

Peripheral blood lymphocyte subclasses were studied by flow cytofluorimetry and monoclonal antibodies in 21 women with Pregnancy Induced Hypertension (PIH), 20 healthy women in their third trimester of pregnancy and in 20 nulliparous, nonpregnant women. The cells were stained with the monoclonal antibodies OKT3, OKT4 and OKT8 to define total T cells, T helper cells (Th) and T suppressor-cytotoxic cells (Ts/c) respectively. B lymphocytes were defined by their surface immunoglobulin. Absolute numbers of total T cells and Ts/c cells were significantly decreased (p less than 0.05) in patients with PIH compared to either control group. The proportion of B lymphocytes was significantly (p less than 0.01) increased and absolute numbers were marginally increased. These findings reflect an immune disturbance which may be of prime importance in the pathogenesis of this disease.

Characteristics of the cell surface antigen, p72, associated with a variety of human tumours and mitogen-stimulated T-lymphoblasts

The monoclonal antibody 791T/36, prepared against a human osteogenic sarcoma cell line, 791T, reacts with a variety of human tumours and also mitogen-stimulated PBMN cells. The target antigen as expressed upon 791T cells is a monomeric plasma membrane-associated glycoprotein with an apparent M r of 72000. By quantitative flow cytofluorimetry, approx. 10 5 antibody molecules bound per cell to T-lymphoblasts induced with PHA or Con A, whereas only a few thousand antibody molecules bound per cell to unstimulated cells, so that the antigen may be classified as a lymphocyte activation antigen. On lymphoblasts, the 791T/36 again reacted with a protein with an apparent M r of 72000. This antigen therefore has a dual role as a tumour marker and lymphocyte activation antigen which may be implicated in the regulation of cell proliferation.

The use of a fluorescent methotrexate probe to monitor the effects of three vinca alkaloids on a mixed population of parental L1210 and gene-amplified methotrexate-resistant cells by flow cytometry.

Cells resistant to methotrexate (L1210/R7A) and possessing an increased level of dihydrofolate reductase due to gene amplification can be detected by the technique of flow cytofluorimetry using a new fluorescent derivative of methotrexate (F-MTX) based on a putrescine linker. Comparative studies of dihydrofolate reductase enzyme and cell growth inhibition following treatment with methotrexate and F-MTX suggest that the two agents possess similar modes of action. In an artificially mixed population of cells sensitive and resistant to methotrexate it is possible, using F-MTX, to recognise and separate distinct cell subpopulations showing differential fluorescence using a fluorescence-activated cell sorter (FACS IV). The selective removal of the resistant cells within a mixed population of sensitive and resistant cells has been demonstrated for 5 X 10(-8) M vinblastine by means of flow cytometry. The effectiveness of the vinca alkaloids decreases in the order vinblastine greater than vindesine greater than vincristine, which previously was shown to be the order of effectiveness in producing collateral sensitivity.

The proliferation kinetics of pulmonary alveolar macrophages.

In this study the proliferation kinetics of pulmonary alveolar macrophages (PAMs) are determined using [3H]thymidine labeling, flow cytofluorimetry, metaphase arrest, and the percentage of labeled mitoses. The demonstration of in situ PAM proliferation is demonstrated beyond doubt. Comparison of the turnover time, calculated from cell kinetic parameters, with the experimentally determined migration times shows that the contribution proliferating PAMs make to their own population size is significant. Indeed, a strong case may be made to show that some 70% of the PAM population is produced by intra-alveolar divisions of "free" PAMs.

Transfer of liposome encapsulated carboxyfluorescein to isolated nuclei

The interaction between phosphatidylcholine vesicles and isolated rat liver nuclei has been examined by studying the uptake of the fluorophore carboxyfluorescein. The kinetics of transfer of the dye, analyzed by flow cytofluorimetry with a Fluorescence Activated Cell Sorter (FACS IV), indicate an efficient delivery to the nucleoplasm. The results reflect a liposome-nuclear membrane interaction which may contribute to the processes which underlie our previously described morphological and functional changes in isolated nuclei treated with phospholipids.

Flow cytometric detection of histamine binding to lymphocytes.

Using flow cytofluorimetry histamine binding was demonstrated by 90-100% of human peripheral blood lymphocytes. Cells were labelled with fluorescein isothiocyanate-human albumin-histamine (FITC-HA-His) and analysed using a Fluorescence Activated Cell Sorter (FACS-II). A parallel control FITC-HA conjugate and blocking with both free histamine and histamine bound to albumin were used to establish specificity. The binding of histamine could be blocked with both H1 and H2 type histamine antagonists. Nylon wool separated T and B lymphocyte subpopulations showed the same high proportions of histamine binding cells.

Monocyte markers and the common acute lymphoblastic leukemia antigen on chronic lymphocytic leukemia cells.

The mononuclear cells from 42 patients with chronic lymphocytic leukemia (CLL) were analyzed for surface markers using monoclonal antibodies and flow cytofluorimetry. All 42 CLLs were Ig+HLA.DR+OKT3-OKT4-OKT6-OKT8- and monoclonal with respect to light chain type. Thirty of the 42 tumors expressed the common acute lymphocytic leukemia antigen, and cells from 18 leukemias stained with the antibodies OKM1 and 63D3, which are specific for different antigens characteristic of myeloid cells. Eight of the leukemias failed to stain with PI153, which has been reported to recognize all B-cell CLLs.

Differential agglutination by soybean agglutinin of human leukemia and neuroblastoma cell lines: potential application to autologous bone marrow transplantation.

Normal human bone marrow cells were mixed with radioactively labeled tumor cells from different leukemia and neuroblastoma cell lines, and the cell mixtures were separated by differential agglutination with soybean agglutinin. It is shown that the cell fraction unagglutinated by soybean agglutinin, which was previously found to be capable of reconstituting the hematopoietic system of lethally irradiated recipients, can be purged of tumor cells with varying efficiency depending on the tumor cell expression of soybean agglutinin receptors as detected by flow cytofluorimetry with fluoresceinated soybean agglutinin.

Identification of an anti-human osteogenic sarcoma monoclonal-antibody-defined antigen on mitogen-stimulated peripheral blood mononuclear cells.

An anti-human osteogenic sarcoma monoclonal antibody (mouse IgG2b) termed 791T/36 was found to exert complement-dependent cytotoxicity against phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear (PBMN) cells. This reaction was examined by flow cytofluorimetry using indirect membrane immunofluorescence to detect cell-bound antibody and by measurement of the binding of fluorescein-isothiocyanate-conjugated 791T/36 antibody to cells. The antibody reacted strongly with peripheral blood blast cells induced by PHA, and there was negligible reactivity with resting lymphocytes. Maximum binding was observed after 3 days' culture with PHA, coinciding with maximum DNA synthesis, and this represented of the order of 2 X 10(5) antibody molecules bound per cell. After cell surface radioiodination of PHA-stimulated PBMN cells, detergent lysis and immunoprecipitation of antigen with 791T/36 antibody and Sepharose-protein A, the apparent molecular weight of this antigen was determined to be 72,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This is identical to that of the 791T/36-defined antigen expressed on various osteogenic sarcoma cell lines [3, 17], and by this criterion the antigen is distinguishable from other cell surface markers of activated human lymphocytes.

Effects of ultraviolet irradiation on the cell cycle in normal and UV-sensitive cell lines with reference to the nature of and defect in xeroderma pigmentosum variant

Analysis of the distribution of cells through the phases of the cell cycle by DNA flow cytofluorimetry has been utilized to investigate the effects of ultraviolet (UV)_ irradiation on cell-cycle progression in normal and UV-sensitive lymphoblastoid cell lines. In time-course studies only slight perturbation of DNA distribution was seen in normal cells, or UV-sensitive familial melanoma (FM) lines in the 48 h following irradiation. Xeroderma pigmentosum (XPA) excision-deficient cells showed a large increase in the proportion of cells in S phase 16–40 h post-irradiation. XP variant (XPV) cells were blocked in G 1 and S phases with the complete absence of cells with G 2 DNA content 16–28 h after irradiation. By 48 h post-irradiation the DNA distribution of XPA and XPV cells had returned to that of an unirradiated control. When colcemid was added to the cultures immediately after irradiation to prevent mitotic cells dividing and re-entering the cell cycle, progression through the first cycle after irradiation was followed. UV irradiation did not affect the rate of movement of cells of G 1 into S phase in normal, FM or XPA cells. The proportion of cells in S phase was increased in UV-irradiated cultures in these cell types and the number of cells entering the G 2 + M compartment was reduced. In UV-irradiated cultures of XPV cells a large proportion of cells was blocked in G 1. The rate of accumulation of cells with G 2 DNA content was equal to that of the control with 4 h post-irradiation, thereafter falling below the control. Thus XPV cells in S phase at the time of irradiation complete DNA synthesis to reach G 2 DNA content. However, cells irradiated in G 1 are blocked from entry into S. These results suggest that there is a defect in XPV cells that affects a step prior to the onset of DNA replication.

The presence of NK alloantigens on cloned cytotoxic T lymphocytes.

A panel of sera raised against NK-1.1 and NK-2.1 alloantigens was tested for reactivity against a panel of cloned antigen-dependent CTL lines. By using indirect immunofluorescence and flow cytofluorimetry, weak, but clear and consistent, reactivity was found on all CTL. Concordant with the genetics of NK alloantigens, C57BL/6-derived clones were reactive with anti-NK-1.1 and anti-NK-2.1 sera, whereas CBA-derived clones were reactive with anti-NK-2.1 sera but not with anti-NK-1.1 sera. Cloned CTL lines were also able to partially and specifically absorb the antibodies from NK alloantiserum that reacted with splenic NK cells. These results indicate that cloned CTL lines express at least some of the NK alloantigen determinants present on splenic NK cells and have important implications regarding the relationship of CTL and NK cells.

Quantification by flow cytofluorimetry of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes

A method allowing quantitative analysis of the expression of foreign antigens at the surface of transformed cells is described. Aminoethyl-Sephadex G-25 beads labelled with different amounts of fluorescein isothiocyanate (FITC) were used to calibrate an Epics V cell sorter. The quantity of FITC molecules bound per bead was found to be a linear function of relative fluorescence intensity expressed by channel number for intermediate and high levels of fluorescence and a non-linear function for low levels. The lowest limit of detectable fluorescence was 3400 FITC molecules bound per bead. Using FITC-conjugated monoclonal antibodies (m.Ab.) the number of HLA class I molecules expressed at the surface of murine LMTK − (H-2K k) cells transfected by cloned HLA class I genes was estimated and compared with the amount expressed on human B (Raji) and T (MOLT 4) lymphoblastoid reference cells. Murine transformed cells expressed approximately 3 times less HLA class I determinants per surface unit (μm 2) than Raji cells and 1.4 times less than MOLT 4 cells. Similar results were obtained by Scatchard analysis of the same populations using radiolabelled m.Ab. A detailed analysis of fluorescent cells showed that 5–20% of transformed cells expressed less than 33,000 HLA class I molecules/cell whereas most MOLT 4 cells exhibited at least 280,000 molecules/cell and the majority of Raji cells more than 700,000 molecules/cell. The expression of foreign antigen did not reduce the amount of H-2K k molecules at the surface of transformed cells (mean of 350,000 molecules/cell).

Quantitative analysis of T-lymphocyte subsets in atopic eczema, using monoclonal antibodies and flow cytofluorimetry.

An analysis of T-lymphocyte subpopulations was performed on thirty-eight consecutive patients with atopic eczema, using monoclonal antibodies and flow cytofluorimetry. The total T-lymphocyte and helper T-cell numbers did not differ significantly from those of a matched control group, but there was a highly significant reduction in suppressor T-cell numbers among the atopic group. Both suppressor T-cell deficiency and circulating IgE elevation were found to correlate positively with disease activity. Food specific IgE levels to milk and egg were ascertained by radioallergosorbent (RAST) studies. These proved positive in seven out of thirty-five atopics (20%).

T helper lymphocyte depression in early human pregnancy

Peripheral blood lymphocyte subclasses were determined in 60 women with normal pregnancies, 20 from each trimester, and in 20 controls using automated flow cytofluorimetry. The cells were stained with the monoclonal antibodies OKT3, OKT4 and OKT8 to stain total T cells, T helper and T suppressor-cytotoxic lymphocytes, respectively. A polyvalent rabbit anti-human Ig serum was used to stain B lymphocytes. Absolute numbers of T lymphocytes were significantly reduced in both the first and second trimesters. This was due to a significant decrease in T helper lymphocytes and a smaller, statistically not significant, reduction in the number of T suppressor lymphocytes. There was no significant change in lymphocyte subclasses during the third trimester. Total lymphocyte numbers were normal throughout pregnancy.

Selection of optical filters for laser flow cytometry using fluorescent conjugates.

AbstractHigh performance optical filters improve sensitivity of detection and resolution of fluorescence in flow cytofluorimetry. Optimal optical barrier filters for laser‐illuminated flow cytofluorimeters differ from those for fluorescence microscopes with wide spectrum illumination. Filters for laser excitation must stop intense illumination at particular wavelengths while transmitting fluorescence emission maximally.Using a Becton, Dickinson Fluorescence Activated Cell Sorter (FACS II) (Becton, Dickinson and Co., Sunnyvale, CA) a range of optical filters were compared for transmission characteristics and performance in fluorescence analysis of cells stained with fluorescein‐ or rhodamine‐labeled antibodies and of green and red fluorescent beads. Interference filters were generally superior to dyed glass filters: the Ortho 528 nm band pass (BP) filter (Ortho Instruments, Westwood, MA) (originally a Schott and Genossen product) was excellent for fluorescein analysis using the 488 nm laser line. In the absence of satisfactory interference filters, a Schott and Genossen 550 nm long pass (LP) filter (Schott America, NY) (dyed epoxy between two glass plates) was best for rhodamine analysis using the 514.5 nm line. Intrinsic fluorescence of some dyed glass filters rendered them unsuitable unless used in a prefiltered light path. Simultaneous analysis using two fluorochromes with overlapping excitation and emission spectra, e.g. fluorescein and rhodamine, will be suboptimal even with electronic dual fluorescence compensation; optimal analysis requires dual laser excitation with geometric separation of the light paths. For single laser systems, sensitivity and resolution are improved by using fluorochromes with fullyseparated emission spectra.

An automated assay of merozoite invasion of erythrocytes using highly synchronized Plasmodium falciparum cultures.

Plasmodium falciparum cultures were synchronized using three lytic treatments with sorbitol. Schizonts from these cultures were used in a rapid, highly sensitive assay of invasion of erythrocytes by merozoites. The parasitaemias in recipient cells after invasion were determined by flow cytofluorimetry after staining with the dye 33258 Hoechst. Invasion of erythrocytes was shown to be reduced by serum from a patient with malaria. The assay is suitable for rapidly screening large numbers of samples, such as monoclonal antibodies.

Study of low density lipoprotein interaction with platelets by flow cytofluorimetry

FEBS LettersVolume 137, Issue 2 p. 319-322 Full-length articleFree Access Study of low density lipoprotein interaction with platelets by flow cytofluorimetry A.V. Mazurov, A.V. Mazurov USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorS.N. Preobrazhensky, S.N. Preobrazhensky USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorV.L. Leytin, V.L. Leytin USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorV.S. Repin, V.S. Repin USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorV.N. Smirnov, V.N. Smirnov USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this author A.V. Mazurov, A.V. Mazurov USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorS.N. Preobrazhensky, S.N. Preobrazhensky USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorV.L. Leytin, V.L. Leytin USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorV.S. Repin, V.S. Repin USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this authorV.N. Smirnov, V.N. Smirnov USSR Cardiology Research Center, Academy of Medical Sciences, Petroverigsky per., 10, Moscow 101837, USSRSearch for more papers by this author First published: January 25, 1982 https://doi.org/10.1016/0014-5793(82)80375-XCitations: 26AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume137, Issue2January 25, 1982Pages 319-322 ReferencesRelatedInformation

Uptake of hematoporphyrin derivative and sensitized photoinactivation of C3H cells with different oncogenic potential

Four types of mouse embryo fibroblast cells of different oncogenic potential were investigated with respect to uptake of the tumor localizing agent hematoporphyrin derivative (Hpd) and sensitized photoinactivation. Cell size and cellular content of Hpd were measured simultaneously for single cells by means of flow cytofluorimetry. The more malignant cell types had a slightly higher porphyrin uptake per unit cellular volume than the untransformed type, while the photosensitivity of cells incubated with Hpd was equal for all the cell types. Since Hpd seems to concentrate in membranes, this may be related to the fact that the membrane areas of malignant cells are relatively larger than that of untransformed cells, due to the presence of more microvilli. The present study indicates that the preferential localization of Hpd in tumors, as well as the high efficiency of phototherapy reported in the literature, are due to extracellular differences between the tumors and normal tissue.

Pharmacokinetics of vinblastine in rat blood plasma and tumour tissue determined by a biological method using flow cytofluorimetry

For the optimal use of vinblastine as an antineoplastic agent, knowledge of the pharmacokinetic distribution in blood plasma and tumour tissue is necessary. A method is presented for the determination of mitotic inhibitory activity of vinblastine and its metabolites, extracted from tissue samples. With this method concentrations as low as 0.005 mg/l which are biologically active can be reproducibly determined. The method has been applied to follow the concentration of vinblastine in blood plasma and tumour tissue after injection of 1.5 mg of vinblastine/ kg body weight in WAG/Rij rats with a R-1, M tumour of 0.3–1.0 g growing in the flank. The high concentration in plasma immediately after i.v. injection decreases quickly within 24 hr to the minimal detectable concentration in plasma of 0.01 mg/l. Although after i.p. injection the concentration in plasma is lower initially, at 24 hr it tends to be higher than after i.v. injection. In tumour tissue the maximal concentration is reached at 6 hr after i.v. injection ( 3.0 mg/kg) and at 12 hr after i.p. injection ( 0.6 mg/kg). From 12 hr after i.p. injection the concentration remains constant during 2–3 days while after i.v. injection a slow decrease is observed. These pharmacokinetic data are compared with the changes of cell proliferation kinetics in the tumour induced by vinblastine. The finding that after i.v. injection the maximal concentration in tumour tissue is higher and is reached earlier than after i.p. injection correlates well with the higher degree of accumulation of cells in mitosis.

Measurement of daunorubicin uptake of Yoshida sarcoma cells in culture using flow cytofluorimetry

A comparative measurement of the transport and localisation of daunorubicin into Yoshida sarcoma cells, was undertaken by a biochemical extraction process and a flow cytometric method. An advantage of this latter procedure would be to identify subpopulation of cells which have enhanced or impaired daunorubicin incorporation as well as the ability to exclude any non-specific incorporation into cell debris, which would otherwise interfere with the overall estimation. It has been possible to use the Biophysics argon ion laser at a wavelength of 488 nm which coincides with the visible absorption bands of daunorubicin and doxorubicin (adriamycin) and the cytofluorimetric estimations of daunorubicin incorporation have now been compared with biochemically determined uptake in Yoshida cells. A high lethal dose of 10 μM was required to achieve the direct measurement by cytofluorimetry procedures on the Biophysics instrument. From cell fractionation and CHCl 3/amyl alcohol extraction, it was possible to show that during a 5-h exposure period to daunorubicin (10 μM), the uptake into the nucleus was at first rapid and that into the cytoplasm was much slower. After about 3-h incubation, the level in the cytoplasm decreased, followed by a decrease from the nucleus 1 h later. This could be equated when observed microscopically to the gain in fluorescent cell debris. If all nuclear binding is to DNA, then at the level of (10 μM) concentration in the medium, the number of base pairs to daunorubicin would be 9 : 1, respectively. Cytofluorimetry showed a broad spread of intracellular daunorubin fluorescence which increases with cell size. Increasing external concentration caused a more rapid incorporation as well as a quicker release from the cells.