Flow Cytometric Cell Sorting Research Articles

Isolated human follicular dendritic cells display a unique antigenic phenotype.

In the present study, follicular dendritic cells (FDCs) were purified to homogeneity in order to define the lineage and function of these cells. FDCs were identified by their characteristic morphology and by their expression of receptors for the third complement component, the myeloid-restricted antigen CD14, and the FDC antigen DRC-1. Unclustered FDCs displayed a unique antigenic phenotype since they expressed several B- and myeloid lineage-restricted antigens, but lacked T and NK cell antigens as well as the leukocyte common antigen. FDCs expressed adhesion molecules, including most of the VLA proteins, intercellular adhesion molecule 1 (ICAM-1), and CD11b. FDCs could be isolated to homogeneity by their intense staining with anti-CD14 using flow cytometric cell sorting. These highly purified FDCs expressed CD14 and CD21 but lacked CD20. This antigen pattern and characteristic morphology confirmed that these cells were, in fact, homogeneous FDC preparations. Analysis of polymerase chain reaction-amplified cDNA from highly purified FDCs showed no transcripts for IL-6. The isolation of homogeneous FDC populations will be important for the analysis of the functional role of FDCs within the lymphoid follicle.

Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting.

To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrug-resistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrug-resistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window. Net growth of cells in culture was observed 15-20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160-kD membrane species identified by Vinca alkaloid photoaffinity labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of bronchoalveolar lavage cells and macrophage-derived chemoattractant activity in pancreatic elastase-induced emphysema in hamsters.

Bronchoalveolar lavage (BAL) was performed 30 days after endotracheal saline (C) or pancreatic elastase (E) administration to hamsters. E animals had twice as many cells as C animals (7.04 +/- 0.07 vs. 6.69 +/- 0.05 log10 cells, p less than 0.0001, paired t-test) and greater numbers of polymorphonuclear neutrophils (PMN) (6.61 +/- 0.05 vs. 5.27 +/- 0.04 log10 cells/animal, p less than 0.0001, t-test). On flow cytometric analysis and cell sorting, BAL cells from C animals showed a homogeneous population of cells with high light scatter and laser-excited intrinsic autofluorescence that were predominantly (greater than 99%) pulmonary alveolar macrophages (PAM). BAL cells from E animals had 80% PAM with characteristics similar to those of C animals; the remaining 20% of cells were smaller with lower forward and 90 degrees light scatter and four- to fivefold lower autofluorescence. These were found to be predominantly PMN on cytological examination of a sorted fraction. Fc receptors were expressed by greater than 95% of BAL cells from both C and E animals. To explain the altered cell population in E animals, PAM from both E and C animals were cultured in vitro, and the supernatants were assayed for neutrophil chemoattractant activity (NCA). In vitro PAM from E animals produced greater NCA than control PAM after 48 h in culture when stimulated with aggregated immunoglobulin or serum activates zymosan (p less than or equal to 0.003, ANOVA). Analyses of BAL supernatants 24 h after elastase administration indicated the presence of a heat-stable chemoattractant for PMN; such chemotactic activity, however, was not detectable 1 week or 4 weeks after the administration of elastase.

Effects of Flow Cytometric Analysis and Cell Sorting on Photosynthetic Carbon Uptake by Phytoplankton in Cultures and from Natural Populations

Photosynthetic rates of phytoplankton were significantly lower after analysis by flow cytometry (FCM) than before. Exposure to the laser beam during the sorting process caused significant physiological damage. The cellular content of a radiolabel accumulated prior to FCM was not affected by FCM. Although it may not be possible to use FCM to preconcentrate cells for further physiological studies, samples may be incubated with stable or radioactive isotopes and then analyzed by FCM.

Growth kinetics as a function of ploidy in diploid, tetraploid, and octaploid smooth muscle cells derived from the normal rat aorta.

The smooth muscle cell population in major arteries of humans and experimental animals is heterogeneous with regard to cellular DNA content. A proportion of cells has polyploid DNA content and this proportion increases with normal aging and with hypertension. We have isolated pure populations of rat aortic smooth muscle cells containing 2C, 4C, and 8C DNA content by cloning of cultures of cells previously subjected to flow cytometric cell sorting. Karyologic analysis of these clonal populations revealed them to be pure diploid, tetraploid, and octaploid populations, respectively, containing 2N (= 42), 4N, and 8N chromosomes. Cell attachment area and nuclear size appeared to increase with the level of ploidy. Studies of the proliferative characteristics of the cells revealed that the growth rate and ultimate cell densities achieved decreased as the ploidy level increased. The intrinsic cellular radiosensitivity of these clones did not vary with ploidy. Increased smooth muscle cell ploidy is, therefore, associated with a decreased rate of proliferation. The emergence of smooth muscle cells with polyploid DNA content under normal and pathologic conditions is probably due to mitotic polyploidization without net cell proliferation and may be related to the need for expression of differentiated functions.

Isolation and functional analysis of human B cell populations. I. Characterization of the B1+B2+ and B1+B2- subsets.

Distinct populations of human B lymphocytes can be identified by their expression and/or co-expression of the B cell-restricted antigens B1 and B2. Dual fluorochrome staining and flow cytometric cell sorting permitted the isolation of the B1+B2+ and B1+B2- cells to homogeneity. In contrast, very few B1-B2+ cells were obtainable from normal lymphoid organs. Virtually all B1+B2+ cells expressed IgM and IgD, but lacked IgG and the plasma cell antigens PCA-1 and PC-1, whereas the B1+B2- cells more frequently expressed IgG, PCA-1 and PC-1. Both populations were noncycling and were composed of similar percentages of small and large cells. The B1+B2+ cells proliferate to anti-mu or to anti-mu + PHA-LCM, but not to PHA-LCM alone. They require both T cells and PWM to produce Ig. In contrast, B1+B2-cells do not significantly proliferate to anti-mu, PHA-LCM, or anti-mu and PHA-LCM. They produce Ig in response to T cells alone without PWM. These phenotypic and functional observations provide preliminary evidence that these populations are distinct and that the B1+B2+ cell may be a "resting" B cell, whereas the B1+B2- cell appears to be more "differentiated." The present studies further suggest that they will also be helpful in characterizing B cells in some human disease states. We believe that the identification and isolation of these and similar subsets of B cells defined by differing cell surface phenotype should aid our understanding both of normal B cell differentiation and of B cell disease states.

Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta.

Smooth muscle cells with 4C (double diploid) DNA content have been found in major arteries. The proportion of 4C cells increases with normal aging and with hypertension. These cells may represent a state of arrest at the G2 phase of the cell cycle or may be examples of true tetraploidy. Flow cytometric cell sorting was used to isolate 4C smooth muscle cells from the rat aorta, and the cells were cultured. Flow cytometry, Feulgen microdensitometry, and karyotyping of the progeny of the 4C cells established the presence of true tetraploid cells. These findings demonstrate the presence of reproductively viable tetraploid cells in a normal mammalian tissue.

Stable quantitative differences of antigen expression in human melanoma cells isolated by flow cytometric cell sorting.

The stability of expression of a membrane antigen on human FME melanoma cells was investigated by means of flow cytometric cell sorting and analysis. The melanoma-associated 250 kd antigen was strongly expressed on all cells, as recognized by binding of the monoclonal antibody 9.2.27. By flow cytometric cell sorting, cells of high and low antigen expression were isolated, and the difference in antigen expression between the two populations was examined as a function of time in culture. Immediately after sorting, the median fluorescence intensities of the two populations differed by a factor of 2.7. After the first few days in culture, much of the range in antigen expression of the parent population was regenerated. However, a lasting difference in antigen expression was established, corresponding to 50% higher density of antigen on the cells sorted for high fluorescence intensity, compared to those sorted for low intensity. After trypsin treatment, which removed the antigen from the cell surface, normal antigen expression was regained after 2-3 days in culture, with the same difference between the two populations as before the trypsin treatment. The stability of the established difference in antigen expression between the two sorted subpopulations indicates that expression of this antigen is a precisely controlled, heritable characteristic of the FME melanoma cells.

An ultra-pure in vitro phase synchrony method employing centrifugal elutriation and viable flow cytometric cell sorting.

We present a method of synchronizing cells in G1-, S-, and G2M-phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst-33342 stained Chinese hamster ovary cells. G1- and S-phase cells can be separated to greater than 99% homogeneity and G2-M to 70% purity. Most of the 30% contamination in the G2-M fraction was due to S-phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H-TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.

Coulter Volume Cell Sorting to Improve the Precision of Radiation Survival Assays

A new method for measuring cell survival at low doses of ionizing radiation has been developed through the use of flow cytometric cell sorting on the basis of Coulter volume signals. The cell sorter is capable of deflecting a precisely known number of cells directly into culture dishes, thus eliminating any errors associated with cell dilution and volume sampling. The use of Coulter volume signals as the sorting parameter is shown to be noncytotoxic for a variety of cell lines. Comparison of radiation survival curves measured above the 10% survival level by either the cell sorter or standard dilution assay demonstrates the increased precision of the cell sorter technique . Because of these advantages of cell sorting over conventional methods of plating cells, this technique has many applications in the field of radiation biology and other studies of cell survival.

Protein synthesis as a function of protein content in exponentially growing NHIK 3025 cells studied by flow cytometry and cell sorting

Methods of cell preparation and quantitative flow cytometric cell sorting were developed for precise measurements of the incorporated radioactivity in cells labelled with [ 14C]-valine and fractionated as a function of their fluorescence intensity after staining with fluorescein-iso-thiocyanate (FITC). FITC fluorescence intensity of exponentially growing NHIK 3025 cells was found to be directly proportional to cellular protein content as measured by saturation-labelling with [ 14C]-valine. Protein synthesis as measured by 14C incorporation after pulse-labelling was found to increase nearly proportionally with cellular protein content. The deviations from proportionality were not greater than 6%, but yet found to be significant since the measurement error corresponded to only 2% standard deviation.