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PubMed1984

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.

Animals↗
PubMed2000

Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent.

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.

Animals↗
PubMed1995

Establishment and evaluation of MRK16-magnetic cell sorting assays for detecting low expression of multidrug resistance P-glycoprotein using human leukemia cell lines and peripheral blood cells from healthy donors.

Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established to detect low expression level of P-glycoprotein (P-gp) using a monoclonal antibody MRK16, which recognizes a cell surface epitope of P-gp. With K-562 and U-937 cell lines, which are known to express low levels of P-gp and hence routinely used as negative control cell lines in conventional flow cytometry, both assays gave significantly positive reactivities indicating improved specificity and sensitivity of these assays. The findings in the dilution test, where P-gp-positive cells were added to P-gp-negative cells at various ratios, demonstrated that the MRK16-MACS assay is quantitative and capable of detecting small numbers of P-gp-positive cells as few as 2.5% of the total cells tested. Furthermore, specific enrichment of P-gp-expressing cells in magnetic cell sorting assays was verified by reverse transcription-polymerase chain reaction (RT-PCR) analysis and functional assay for P-gp with Rhodamine 123. The availability of such magnetic cell sorting assays may offer an approach to quantitate low level of P-gp expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1979

Plasmodium berghei - infected red cells sorted according to DNA content.

A cell-sorting method is described for the analysis and separation of red blood cells in Plasmodium berghei-infected mouse blood based on their DNA content. This method involves a selective uptake of the bis-benzimidazole dye 33258 Hoechst, a DNA-binding dye, by red blood cells containing parasites. Infected blood is incubated at 37 degrees C with the dye then washed at 4 degrees C to remove unbound dye. Uninfected cells are then non-fluorescent at the characteristic wavelengths for 33258 Hoechst excitation and emission, whereas parasitized cells display fluorescence intensities in approximately direct proportion tothe number of parasite nuclei (i.e. amount of parasite DNA) within the cell and can be sorted accordingly. Providing cells were incubated in a complex nutrient medium during dye uptake at 37 degrees C, the sorted parasite-infected cells produced lethal P. berghei infections when injected into BALB/c mice. The dye-labelling technique is simple and sufficient red blood cells at various stages of infection can be collected for biochemical or immunochemical studies by cell sorting.

Animals↗
PubMed1994

Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption ("panning") and fluorescence-activated cell sorting.

We developed methods for enriching fetal hepatoblasts by combining panning and multiparametric fluorescence-activated cell sorting. In unpurified, dissociated fetal liver cell suspensions of embryonic age day 15, 3.2% +/- 1.3% and 2.5% +/- 0.7% cells expressed albumin and alpha-fetoprotein, respectively. The remainder exhibited a hemopoietic, endothelial or stromal cell phenotype. Cells were panned first with an antibody to red blood cells to remove erythroid cells and then with monoclonal antibodies OX-43/OX-44 to remove hemopoietic and endothelial cells. This procedure eliminated 84% of fetal hepatic cells, with enrichment of the remainder for albumin or alpha-fetoprotein expression (up to sixfold increase). Flow cytometric analysis of unlabeled cells revealed two populations, which differed in granularity and autofluorescence. After panning, fluorescence-activated cell sorting for agranular cells yielded OX-43/44-positive cells that were essentially all hemopoietic precursor cells or OX-43/44-negative cells that were mostly hemopoietic precursor cells, along with 3.0% +/- 0.7% alpha-fetoprotein-positive cells. In contrast, sorting for granular, OX-43/44-negative cells enriched for predominantly alpha-fetoprotein-positive, parenchymal precursor cells (75.1% +/- 4.7%). Multiparametric flow cytometric analysis of the expression of an oval cell antigen, OC.3, which is a bile duct and putative liver stem cell marker, showed that most OC.3-positive cells coexpressed OX-43/OX-44 and morphologically were hemopoietic precursor cells. However, approximately 30% of the OX-43/44-negative, granular cells expressed OC.3. Although the physiological significance of OC.3-positive hepatoblasts remains to be determined, the ability to isolate distinct liver cell populations by means of fluorescence-activated cell sorting should facilitate further studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1999

Detection of circulating melanoma cells by immunomagnetic cell sorting.

We developed a cellular approach to the identification of circulating melanoma cells in peripheral blood using immunomagnetic cell sorting. One hundred seventy-eight blood samples from 129 melanoma patients and 30 samples from healthy persons and nonmelanoma patients were examined. After density gradient centrifugation the interphase was incubated with the mAb 9.2.27. Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting. Cells were stained using an alkaline phosphatase-antialkaline phosphatase assay and examined by light microscopy. In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per ml whole blood could be detected reliably with the assay. Circulating melanoma cells were not found in 30 controls examined, nor were 9.2.27-positive cells found in 41 patients with primary malignant melanoma. In patients with regional lymph node metastases and in patients with disseminated disease, circulating 9.2.27-positive cells could be detected in 3 out of 22 patients (13.6%) and 10 out of 66 patients (15.2%) examined. We present a sensitive and specific immunocytological approach to detect circulating melanoma cells in peripheral blood. The method is not suitable for early detection of metastases but is a valuable tool for further investigating biological characteristics of circulating melanoma cells.

Antibodies↗
PubMed1972

Do morphogenetic tissue rearrangements require active cell movements? The reversible inhibition of cell sorting and tissue spreading by cytochalasin B.

Previous studies have indicated that cell sorting and tissue spreading are caused by cell combination-specific differences in intercellular adhesive energies, acting in a system of motile cells. We wished to determine whether these adhesive energies could drive cell rearrangements as well as guide them. Accordingly, aggregates of intermixed embryonic cells were cultured in solutions of the drug cytochalasin B (CCB) at a concentration shown to inhibit the locomotion of cells on a solid surface. In addition, spherical aggregates of several kinds were cultured in mutual contact under similar conditions. Both cell sorting and tissue spreading were found to be inhibited. The prompt release of this inhibition upon removal of the CCB showed that the inhibited cells were not merely injured. Moreover, aggregation experiments showed that CCB did not prevent cells of several kinds from initiating mutual adhesions. In fact, heart cell aggregation was enhanced by CCB. We conclude that interfacial forces, originating outside the cell, act together with forces originating inside it in bringing about the morphogenetic movements of cell sorting and tissue spreading. We propose the term "cooperative cell locomotion" to describe translational movements of cells arising from such a combination of intrinsic and extrinsic forces.

Animals↗
PubMed1999

Cell-sorting in aggregates of Dictyostelium discoideum.

When Dictyostelium cells are induced to develop between a coverslip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three-dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion.

Animals↗
PubMed1994

Magnetic activated cell sorting (MACS)--a new immunomagnetic method for megakaryocytic cell isolation: comparison of different separation techniques.

Megakaryocytes are difficult to isolate because of their fragility, their tendency to aggregate, and their varying sizes. For purification of cells at different stages of maturation and of different sizes (ploidy classes) we developed an immunomagnetic cell sorting method (MACS) to enrich the whole spectrum of the megakaryocytic cell lineage. The use of small magnetic beads coupled to various antibodies and labelling with fluorescent antibodies allowed direct analysis of enrichment and evaluation of the isolated fraction without further staining or detachment procedures. CD 61 (Y2/51), a monoclonal antibody directed against platelet glycoprotein IIIa, was employed to perform the separation procedure. An enrichment up to 47% of CD 61-positive cells with an average of 37% and a recovery rate of 37% was obtained by using the MACS technique. Pre-enrichment by Percoll density centrifugation, followed by MACS separation, resulted in an enrichment of 65% and a recovery rate of 67%. The relative amount of small megakaryocytic cells in only MACS-enriched cell populations, however, was higher than in Percoll/MACS fractions. As a parameter of vitality we tested cytokine secretion of the enriched megakaryocytes in reverse haemolytic plaque assays. Secretion of IL-1, IL-6, GM-CSF, and PDGF with and without stimulation by phorbol myristate acetate was demonstrable at the single cell level.

Bone Marrow Cells↗
PubMed1980

Vital DNA staining and cell sorting by flow microfluorometry.

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.

Amidines↗
PubMed1998

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting.

Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.

3T3 Cells↗
PubMed1988

[Cell sorting in flow cytometry].

The aim of the presentation is to introduce a dramatic accomplishments of high technology and the current applications of medicine, particularly in the field of Gynecology and Obstetrics. An author's demonstration includes both of a brief basic concept of window and fraction sorting and some clinical applications. Cell sorting based on genotypic (neoplastic) and phenotypic markers of a benign and malignant lesions of female genital tract seems to be expressed at the cellular and molecular level in terms of degree of cell growth, differentiation and functions, which are not readily appreciated by descriptive morphology. DNA aneuploidy is a conclusive marker of malignant characteristics and a monoclonal antibodies of Bromodeoxyuridine (BrdU), an analog of thymidine, is an available identification of DNA synthetic cells. Among phenotypic cell markers, surface membrane antigens have been extensively studied on the immunological state in pregnancy, peripheral lymphocyte subsets from pregnant women by the use of monoclonal antibodies. From gynecological and obstetrical patients management perspective, a role of cell sorting technology in flow cytometry is emerging as a tool for diagnosis of cancer (DNA abnormalities and cell cycle analysis) multiparameter analysis in quantitation (DNA base pairs, RNA and protein), surface markers for immunological state in pregnancy (membrane antigen and monoclonal antibodies), prognosis (adverse impact of aneuploidy and high S percentage using BrdU with monoclonal antibody) and targeting cytotoxicity to steroid responding tumor cells (estrogen receptor and site-directed chemotherapy).

Aneuploidy↗
PubMed1983

Analysis and separation of murine bone marrow stem cells by H33342 fluorescence-activated cell sorting.

Murine bone marrow cells were stained with the fluorescent bisbenzimide dye H33342, a supravital DNA stain, and sorted on the basis of differences in fluorescence intensity by a light-activated cell sorter. Sorted cells were submitted to assays to enumerate spleen colony forming cells (CFUs), HPP-CFC and GM-CFU-1 and -2. The recoveries of these cell types after the separation was 70 to 120%, except for GM-CFU-2 (10-40%). The frequency distribution of these cell types with respect to their fluorescence intensity suggested that the majority of CFUs and HPP-CFC are quiescent, whereas GM-CFU-2 are proliferating. Furthermore, HPP-CFC could be separated from GM-CFU-1 and -2 on the basis of fluorescence intensity differences, which indicates that these cell types are different. CFUs determined 10 days after irradiation and grafting of the recipient mice were distributed evenly over the two fluorescence intensity subpopulations which contained most of the HPP-CFC and the GM-CFU. The same was observed at day 8. However, CFUs determined at day 12 were only present in the subpopulation of low fluorescence intensity which also contained most of the HPP-CFC. This observation provides evidence for heterogeneity of the spleen colony forming cells.

Animals↗
PubMed1978

Studies on dispersed unincubated chick blastoderm cells. II. Formation of contacts and cell sorting in aggregates of unincubated chick blastoderm and heart cells.

The configuration of blastoderm-heart cell aggregates after cell sorting depended largely on the proportion of each cell type initially present in the culture. In heterotypic aggregates, blastoderm cells were always found to partially or completely surround heart cells with both cell types retaining their characteristic morphology. Specialized junctions (e.g. desmosomes) developed only between cells of the same type. These observations suggest that blastoderm cells are unable to recognize and form stable contacts with heart cells.

Animals↗
PubMed1993

Magnetic affinity cell sorting (MACS) separation and flow cytometric characterization of neural cell adhesion molecule-positive, cultured myogenic cells from normal and dystrophic dogs.

We developed a magnetic affinity cell sorting (MACS) assay based on differential expression of neural cell adhesion molecule (NCAM) isoforms in muscle cell cultures from normal and dystrophic dogs. NCAM is expressed during normal muscle differentiation, but has not been extensively examined within the context of muscle disease. A myogenic MACS assay could potentially maximize chances of obtaining normal nonsenescent, low-passage myogenic cells capable of proliferating in vitro and in vivo following transplantation. Myoblast-specific anti-NCAM polyclonal antibody directed against the NCAM isoform associated with muscle cell proliferation more effectively separated mixed canine cultures than did monoclonal antibodies directed against differentiated NCAM isoforms in the MACS assay. Flow cytometry using 5.1H11 anti-NCAM monoclonal antibody was then performed on normal and dystrophic fractionated cells and the results from these two groups were compared to each other and to nonfractionated cell populations. Normal canine cell cultures that had not been separated contained a larger percentage of FACscan-positive cells than did corresponding dystrophic canine cell cultures. Prior polyclonal anti-NCAM MACS separation of dystrophic cultures yielded higher numbers of adherent cells and higher gating percentages of 5.1H11-positive cell populations than did normal cultures. However, cells from dystrophic animals exhibited lower mean fluorescent expression of NCAM than normal cells.

Animals↗
PubMed1977

Isolation of glial cell-enriched and -depleted populations from mouse cerebellum by density gradient centrifugation and electronic cell sorting.

Preparative amounts of populations enriched and depleted in glial cells have been isolated from 10-day-old mouse cerebella. Discontinuous density bovine serum albumin (BSA) gradients provide two distinct populations: the one derived from the 10-15% BSA interface is enriched in cyclic nucleotide phosphohydrolase (CNPase) activity, and a percentage of S-100, GFA protein, and NS-1 antigen-positive cells; the other, located as the 15-31% interface, contains a cell population depleted in these compounds. This report also describes the first use of flow microfluorimetry and electronic cell sorting techniques for the analysis and isolation of cell populations derived from the mammalian central nervous system. Glial cell-enriched and -depleted populations obtained by BSA density gradient centrifugation were analyzed for their forward angle light scattering properties and their capacity to bind anti-corpus anti-serum to the cell surface. The glial cell-enriched fraction shows a different frequency distribution of light scattering than the glial-depleted fraction. Anti-corpus callosum antiserum binds preferentially to the glial cell-rich fraction. Cells can be sorted into corpus callosum antigen-positive and -negative cell fractions and recovered with a viability of more than 95% as judged by trypan blue exclusion. Anti-corpus callosum-positive sorted cells are enriched in CNPase activity, S-100, and glial fibrillary acidic proteins.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed2001

Automated single-cell sorting system based on optical trapping.

We provide a basis for automated single-cell sorting based on optical trapping and manipulation using human peripheral blood as a model system. A counterpropagating dual-beam optical-trapping configuration is shown theoretically and experimentally to be preferred due to a greater ability to manipulate cells in three dimensions. Theoretical analysis performed by simulating the propagation of rays through the region containing an erythrocyte (red blood cell) divided into numerous elements confirms experimental results showing that a trapped erythrocyte orients with its longest axis in the direction of propagation of the beam. The single-cell sorting system includes an image-processing system using thresholding, background subtraction, and edge-enhancement algorithms, which allows for the identification of single cells. Erythrocytes have been identified and manipulated into designated volumes using the automated dual-beam trap. Potential applications of automated single-cell sorting, including the incorporation of molecular biology techniques, are discussed.

Automation↗
PubMed1988

Expressed recombinant cadherins mediate cell sorting in model systems.

Cadherins are cell-surface glycoproteins responsible for Ca2+-dependent cell-to-cell adhesion. E- or P-cadherin was transfected into L cells, which normally have little cadherin activity, and cellular aggregation of the resulting transfectants was observed to be a function of the cadherin molecule expressed. Transfected cells preferentially adhered to cells expressing the same cadherin subclass. Furthermore, in reconstituted embryonic lung tissue, E-cadherin-expressing L cells were associated with epithelial tubules expressing E-cadherin, while untransfected L cells associated with mesenchymal cells. These results provide the first direct evidence that the differential expression of cadherins can play a role in cell sorting in heterogeneous cell populations.

Agglutination↗