Ontogenetic analysis of some surface markers on pig lymphocytes using fluorescence-activated cell sorter

Surface markers were demonstrated on pig lymphocytes using anti-T cell-IgG and anti-Helix pomatia (HP) IgG during prenatal and postnatal development. A fluorescence-activated cell sorter analysis of T-cell surface markers was accompanied by an image analysis to prove the association of T antigenic determinants with the plasma membrane only. We found development-dependent changes in both anti-T cell and HP surface markers in both primary and secondary lymphatic organs. The number of T-positive (T+) cells estimated by anti-T cell-IgG was very similar to the results obtained by spontaneous E-rosette forming tests. At all selected age intervals, changes in the number of T+ cells were not significant in the thymus, but a marked increase in T+ cells was found in both spleen and lymph nodes. The image analysis confirmed the expression of T cell markers on the cell surface. The distribution of T cell markers was uneven, i.e. various degree of fluorescence intensity on whole ring-pattern projection of the cell surface image was estimated. In second lymphatic organs especially, fluorescence intensity of cells, i.e. total number of T cell markers estimated by anti-T cell-IgG, increased with age. On fetal day 73, T cell markers were slightly expressed, but very high fluorescence intensity and heterogeneous distribution of T cell markers on lymphocytes were found on fetal day 107 and postnatal day 56. The results indicate the possibility of functional maturation of various T cell markers on T cell subsets, furthermore a different degree of expression of T cell markers on various T cell subsets can be suggested. The number of HP+ cells increased with age in both primary and secondary lymphatic organs. In the prenatal period, the expression of HP receptors was very weak in both primary and secondary organs in contrast to the marked increase in HP+ cells during the postnatal interval. Differences in fluorescence intensity of cells were found, representing the increase by 22% in thymus cells comparing to cells of secondary lymphatic organs. Heterogeneity of HP+ cell populations in thymus was shown by the Scatchard plot, indicating at least two subpopulations of HP+ cells with different avidity to HP. Cells with low HP avidity could include a subset with cytolytic activity.     

Preservation of cells sorted individually onto microscope slides with a fluorescence-activated cell sorter

Fluorescence-activated cell sorters permit analyses and separation of cell populations based on light scatter and surface immunofluorescence parameters. Since a sorter can deposit individually identifiable cells onto a microscope slide, it was considered of interest to combine the flow measurements with analyses available on cells adhering to a surface as in, for example, morphological studies, cytoplasmic immunofluorescent staining, and mRNA in situ hybridization. A necessary condition for these studies is the preservation of cell structures after sorting. We report here a procedure suitable for this purpose. The most important features of this procedure are A) reducing the saline content of the sorter sheath fluid to about 0.0015 M (one-hundredth that of normal saline) to prevent cell damage due to hypertonicity during drying, and B) coating the substrate with a thin layer of newborn calf serum to promote the adherence of the cells to the substrate during subsequent fixing and staining.     

Single-cell isolation and cloning of Tetrahymena thermophila cells with a fluorescence-activated cell sorter

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.     

Transfer of mouse IgG2 production by IgM-bearing spleen cells separated by a fluorescence-activated cell sorter.

The purpose of the present study was to determine whether at least some splenic B lymphocytes can switch from the synthesis of one isotype of immunoglobulin to another during B cell differentiation. The experimental system invovled the transfer of characterized cell suspensions between allotype congenic strains of mice followed by analysis in the recipient for donor type immunoglobulin production. Donor splenic lymphocytes were incubated with specific fluorescent labelled anti-mu antiserum and passed through the Los Alamos fluorescence-activated cell sorter; mu-depleted cell suspensions were transferred into sublethally irradiated congenic recipients and the amount of donor type immunoglobulin of IgG2 type was measured at weekly intervals. The results demonstrated taht at least some cell bearing membrane bound IgM can differentiate in vivo into IgG2-secreting cells, although not all IgG2-secreting cells have been recently derived from IgM positive precursors.     

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.     

Alignment and focusing unit for dual-laser excitation in the fluorescence-activated cell sorter

The two laser beams in a dual-laser fluorescence-activated cell sorter FACS-II can be aligned and focused independently on the sample stream with an additional unit, which can be fitted easily on the optical bench of the FACS. The unit consists of two spherical lenses, which have been mounted in separate holders and can be moved in three directions by way of micrometer gauges. The lenses, which have different focal lengths, have been cut off on one side so each laser beam only passes one lens. The setup has been tested using the flow analysis of a suspension of double-stained chicken red blood cells. The histograms of both fluorescence signals showed normal distributions with a coefficient of variation of approximately 6%. After willful interference with the adjustments, the laser beams could be readily readjusted within five minutes.     

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.     

Measurement of lysosomal glucocerebrosidase activity in mouse liver using a fluorescence-activated cell sorter assay

Lysosomal acid beta-glucocerebrosidase hydrolyzes glucocerebroside to glucose ceramide. Patients diagnosed with Gaucher disease, however, lack this enzyme, leading to the accumulation of glucocerebroside in tissue macrophages within multiple organs. Such patients can receive enzyme replacement therapy during which a human placental-derived or recombinant form of acid beta-glucocerebrosidase is targeted to the macrophages. As part of evaluating the effectiveness of such therapies, currently available methodologies for measuring acid beta-glucocerebrosidase activity are primarily conducted in cultured cell lines or tissue culture. However, these in vitro assays are limited by their ability to evaluate the efficacy of in vivo acid beta-glucocerebrosidase replacement therapy in animal models. In particular, there is an unmet need to simultaneously define cellular localization and evaluate enzyme activity following treatment in vivo. In addition, results of commonly used fluorescent-based assays for enzyme activity are difficult to compare day to day and/or across laboratories due to the variability inherent in flow cytometric measurement. In this article, we describe a reproducible and consistent quantitative method for the combined measurement of fluorescein intensity from enzyme-substrate conversion and cell localization by phenotype-specific phycoerythrin-antibody staining. Following infusion of recombinant human acid beta-glucocerebrosidase in mice, nonparenchymal cells are prepared from the livers of treated and control animals. Acid beta-glucocerebrosidase activity is measured in molecules of equivalent soluble fluorophore units within Kupffer cell populations as defined by phenotype-specific monoclonal antibodies. This assay should be applicable to investigations of other Gaucher disease treatments in both human and animal models.     

Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

We have used a mouse mammary tumor virus (MMTV)-infected rat hepatoma cell line as a model system for studying glucocorticoid action. These cells induce tyrosine aminotransferase and MMTV in response to the synthetic glucocorticoid, dexamethasone. The major viral antigen, a glycoprotein of 52,000 daltons (gp52), appears on the surface of infected cells in amounts which reflect the cytoplasmic content of viral RNA. Using an anti-gp52 antiserum and a fluorescence-activated cell sorter (FACS), we have selected variants which display low levels of pg52 in the presence of the hormone. Multiple cycles of enrichment for cells that fluoresce weakly in the presence of hormone have generated a population which fails to produce a detectable increase in cell surface gp52 in response to dexamethasone. This population of nonresponders and a number of independent clones derived from this population were analyzed for their ability to induce gp52 and TAT and for these presence of glucocorticoid receptors. All nonresponder clones exhibited little or no induction of either glucocorticoid-inducible marker. Two of the clones contained reduced levels of glucocrticoid receptor, while the remainder of the clones showed no detectable specific hormone binding. These results provide genetic evidence that a single class of glucocorticoid receptors is involved in the induction of both MMTV and TAT in HTC cells.     

Purification of interstitial cells of Cajal by fluorescence-activated cell sorting

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000–40,000 cells and 60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched 6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested 2,000–4,000 cells from single gastric corpus-antrum muscles and detected an 30-fold increase in c-kit mRNA, no enrichment of mast cells, and an 4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses. mouse; c-kit; macrophage; dendritic cell; mast cell     

Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting

The remarkable capability of planarian regeneration is mediated by a group of adult stem cells referred to as neoblasts. Although these cells possess many unique cytological characteristics (e.g. they are X-ray sensitive and contain chromatoid bodies), it has been difficult to isolate them after cell dissociation. This is one of the major reasons why planarian regenerative mechanisms have remained elusive for a long time. Here, we describe a new method to isolate the planarian adult stem cells as X-ray-sensitive cell populations by fluorescence-activated cell sorting (FACS). Dissociated cells from whole planarians were labeled with fluorescent dyes prior to fractionation by FACS. We compared the FACS profiles from X-ray-irradiated and non-irradiated planarians, and thereby found two cell fractions which contained X-ray-sensitive cells. These fractions, designated X1 and X2, were subjected to electron microscopic morphological analysis. We concluded that X-ray-sensitive cells in both fractions possessed typical stem cell morphology: an ovoid shape with a large nucleus and scant cytoplasm, and chromatoid bodies in the cytoplasm. This method of isolating X-ray-sensitive cells using FACS may provide a key tool for advancing our understanding of the stem cell system in planarians.     

Separation of osteoblast-like cells from bone marrow by fluorescence-activated cell sorting

The purification of the osteoblast-like cells (2-3%) among the bone marrow cells (BMC) of C57BL/6 mice using a specific anti-osteoblast serum and a fluorescence-activated cell sorter is described. The antiserum was raised against osteoblast cells isolated from calvaria from neonatal mice. The majority of the cells of the osteoblast-enriched fraction from bone marrow showed a parathormone-induced increase in cyclic adenine monophosphate but no response to calcitonin. This is similar to the response of osteoblast cells obtained from the calvaria. Electron microscopic studies of the extracellular matrix of cultured osteoblast-like cells purified from bone marrow showed the deposition of apatite crystals within and in close apposition to the vesicles. These findings suggest that the isolated cell population was enriched in osteoblasts. Such a cell system from bone marrow might provide an experimental system for investigating the mechanism of bone formation.     

Evaluation of the human sperm acrosome reaction using a monoclonal antibody, GB24, and fluorescence-activated cell sorter

GB24 is a mouse monoclonal antibody raised against human trophoblast microvilli, which recognizes an antigenic determinant on the acrosomal region of the human sperm head. By indirect immunofluorescence, reactivity of GB24 could not be detected on freshly ejaculated spermatozoa but was strongly positive after sperm permeabilization with acetone. On viable, motile spermatozoa, reactivity appeared after induction of the acrosomal reaction with the calcium ionophore A23187. These results suggest that the antigen recognized by GB24 is present on the inner acrosomal membrane. A quantitative evaluation assay of the acrosome reaction on viable spermatozoa by flow cytometry using GB24 and indirect immunofluorescence is proposed.     

Purification of cybrids by fluorescence-activated cell sorting

A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75-100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.     

Kinetics of the apoptotic response induced by anti-IgM engagement of the B cell receptor is dependent on the density of cell surface immunoglobulin M expression

The effect of B cell receptor (BCR) density on anti-BCR-induced apoptosis was assessed in Ramos cell lines, expressing low, medium, or high levels of surface IgM (sIgM(LO), sIgM(MED), sIgM(HI)). All cells required a 6-mug/ml threshold of anti-IgM to elicit apoptosis. Anti-IgM treatment of sIgM(LO) cells induced growth inhibition and limited dose-independent apoptosis. Anti-IgM treatment of sIgM(MED) cells induced dose-independent death with a 32-h lag. Ligation of the BCR in the sIgM(HI) cells induced rapid apoptosis beginning by 6 h, which was dose-dependent. Secondary crosslinking reagents did not affect apoptosis, and this effect was independent of anti-IgM concentration, time, or sIgM density. These results suggest that the response to BCR engagement strongly depends on the cell surface receptor density.     

Staining and fluorescence-activated cell sorter analysis of human lymphocytes using antibodies to a short, chemically synthesized human IL-2 peptide

A rabbit antibody against a chemically synthesized peptide (p84), encompassing residues 59-72 of mature human interleukin 2 (IL-2), has been shown to react specifically with natural or recombinant IL-2. This antibody was used in immunoperoxidase and immunofluorescence techniques for identification of IL-2-containing cells in human peripheral blood or tonsils. Lymphocytes were stimulated with T-cell mitogens (PHA, PWM), fixed, and incubated with affinity-purified anti-p84 antibody, followed by appropriately conjugated secondary antibodies. FACS analysis demonstrated a low fluorescence intensity in 5 to 15% of unstimulated cells. In contrast, 40-60% of mitogen-stimulated cells were stained at a high fluorescence intensity. Staining was inhibited by preincubating the anti-p84 antibody with the homologous peptide or recombinant IL-2, but not by unrelated peptides. In immunoperoxidase staining, anti-p84 antibody reacted selectively with an enriched T-cell population which was 95% Leu 5+, 80% Leu 3+, and 60% Tac+. Thus, this antibody to a synthetic IL-2 peptide reacts selectively with activated T cells, and may serve, therefore, as a useful tool for visualization and enumeration of IL-2-containing cells in blood and tissues.     

Fluorescence-activated cell sorter (FACS) analysis of rabbit cells

Monoclonal antibodies RB1, RB2, RT1, RT2 and RB3 were prepared against rabbit lymphoid cells by immunization with various fractions of rabbit lymphoid cells. The antigens detected by the antibodies are found on B and T cells in different densities. High proportions of polymorphonuclear and bone marrow cells which do not carry the RABELA and RTLA antigens carry the antigens of the RB and RT series. A subpopulation of appendix sIg-negative, RTLA-negative cells has a relatively high concentration of RT2. In general, B and T cells of the appendix show relatively small differences in the membrane densities of RB and RT antigens.