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PubMed1996

Isolation of micronuclei: high yield by sucrose gradient versus maximum purity by cell sorting.

Micronuclei (MNC) of L929 cells were isolated 72 h after irradiation with 6 Gy for characterization of their DNA content, using gel electrophoresis. A novel method for isolation of MNC based on a sucrose gradient ultracentrifugation was developed. With this efficient method (80% recovery) more than 150 x 10(6) MNC per day could be isolated. The purity was > 99%. However, the low number of main nuclei in the MNC isolate ( < 1%) resulted in a contamination of MNC DNA with about 15% main nucleus DNA, due to the several times higher DNA content of main nuclei. Cell sorting was utilized to maximize the purity by choosing the recommended sorting mode for highest purity. Isolation of MNC with the cell sorter was successful (100% purity), but also time-consuming (1-2 x 10(6) MNC per working day could be isolated) and insufficient (10% recovery). Extraction of DNA of these isolated MNC resulted in less than 1 ng/day. Hence, at least 1 week of cell sorting would be necessary for one electrophoretic run. When employing the sucrose gradient method, 2000 times more DNA of MNC have been isolated. We therefore consider this method as the most efficient way for rapid and low cost isolation of large amounts of purified ( > 99%) MNC without the employment of sophisticated and expensive techniques (cell sorting) and the accompanied knowhow. In contrast, maximum purity but low yields of MNC can be obtained by cell sorting.

Animals↗
PubMed1989

Detection of mRNA in flow-sorted cells.

Cells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P-labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co-induction of IL-1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c-fos in U937 to demonstrate linearity. IL-1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS-induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c-fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.

Flow Cytometry↗
PubMed1983

Involvement of a cell-surface glycoprotein in the cell-sorting process of Dictyostelium discoideum.

Cell sorting among prespore cells and prestalk cells in Dictyostelium discoideum was studied by using fluorochrome-labeled cells in an in vitro assay. Labeled prestalk cells first formed randomly mixed aggregates with unlabeled prespore cells. Then cells began to sort out from each other. About 3-4 hr later, prestalk cells became clustered at one pole of the aggregate. Aggregates deposited on an agar surface underwent morphogenesis and formed migrating slugs within 3 hr. The addition of Fab fragments directed against a cell-surface glycoprotein of Mr 150,000 (gp 150) to the cell mixture completely inhibited the cell-sorting phenomenon. Morphogenesis of such aggregates on agar was also delayed by 5 hr. However, inclusion of Fab fragments directed against the endogenous lectins, the contact site A glycoprotein, or vegetative cells had no detectable effect on cell sorting or morphogenesis of these reconstituted aggregates.

Cell Adhesion↗
PubMed1991

Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling.

We have isolated several Chinese hamster ovary cell lines with temperature-sensitive defects in the recycling of receptors after endocytosis. These cell lines were selected using fluorescence-activated cell sorting for retention of a pulse of labeled transferrin after a chase in the presence of unlabeled transferrin. One of these cell lines, TfT1.11, was selected for further characterization. In TfT1.11 the trapping of transferrin within the cells is paralleled by a loss of cell surface transferrin receptors. Within 4 h after the shift from 33 to 41 degrees C the surface binding of transferrin is reduced to 18% of parental cells at 41 degrees C. The trapping of transferrin and the loss of transferrin receptor from the cell surface are caused by a temperature-conditional 5.5-fold decrease in the initial rate of transferrin recycling. TfT1.11 cells also rapidly lose 89% of their ability to take up alpha 2-macroglobulin after the temperature shift to 41 degrees C. These data indicate that the TfT1.11 cell line has a pleiotropic defect in receptor recycling.

Adenosine Triphosphate↗
PubMed1992

An experience of cell sorting using samples isolated from paraffin-embedded tissue of neuroblastoma.

Flow cytometric DNA content analyses were performed on samples from 72 patients with neuroblastoma. Nuclear suspensions obtained from paraffin-embedded samples were prepared using the method described by Schutte et al. Determination of DNA ploidy was possible in 62 out of 72 patients. DNA diploidy was detected in 23 (37.1%) and DNA aneuploidy was found in 39 (62.9%) of these 62 patients. Cell sorting was performed on two peak areas of the DNA histogram of samples with aneuploidy. The sorted cells of the first peak area were composed of small round cells and were considered to be normal lymphocytes. The sorted cells of the second peak area were composed of relatively large cells with obvious nucleoli and were considered to be neuroblastoma cells. Thus, two cell components could be distinguished by cell sorting. It is concluded that cell sorting may be of benefit in giving detailed information about DNA histograms, even in samples obtained from paraffin-embedded tissue.

Cell Separation↗
PubMed1990

Secretagogue-induced oscillations of cytoplasmic Ca2+ in single beta and alpha-cells obtained from pancreatic islets by fluorescence-activated cell sorting.

Pancreatic islet cells from normal adult rats were purified by autofluorescence-activated cell sorting (FACS). A purification of islet beta-cells (greater than 90%) and alpha-cells (greater than 70%) was obtained after FACS separation. Dual-wavelength microfluorimetry was employed for measurements of cytoplasmic Ca2+ concentration (Ca2+i) in single beta- and alpha-cells. In FACS-purified beta-cells, 11.8 mM glucose induced oscillations of Ca2+i with a frequency of 1 cycle per 2-4 min, whereas 20 mM glucose induced a rapid rise in Ca2+i followed by a sustained elevation in Ca2+i. In contrast, alpha-cells exhibited a decrease in Ca2+i following exposure to 20 mM glucose, and subsequent exposure to 20 mM arginine induced a large increase in Ca2+i with periodic fluctuations. FACS purification of a heterogeneous islet-cell population provides a unique model for characterizing secretagogue-induced changes in Ca2+i in single beta-and alpha-cells.

Animals↗
PubMed1983

An approach to the cloning of cell surface protein genes. Selection by cell sorting of mouse L-cells that express HLA or 4F2 antigens after transformation with total human DNA.

We describe an approach to the cloning of cell surface proteins that is independent of messenger RNA isolation. Mouse Ltk- cells are cotransformed with the thymidine kinase gene from Herpes Simplex Virus and total human DNA. Transformants expressing the human surface antigens of interest are isolated by two selection steps, consisting of treatment with hypoxanthine/aminopterin/thymidine and fluorescence-activated cell sorting. Using this procedure, we isolated seven transformants expressing HLA-A,B,C antigens and 12 transformants expressing the 4F2 antigen. We have so far failed to identify any OKT-10 antigen expressing L-cell transformants. Three independent secondary 4F2 transformants were obtained after identical cotransformation of fresh Ltk- cells with DNA from primary transformants. Analysis of their genome by hybridization with human DNA revealed a shared set of human restriction fragments in all three cell lines. This 32 X 10(3) base-pair segment of DNA codes for the human 4F2 antigen, thereby offering the opportunity to clone the gene. To substantiate this hypothesis, we analyzed the seven HLA-expressing cell lines, and we found that all of them had acquired an HLA-coding sequence concomitant to its expression.

Animals↗
PubMed2000

Single cell sorting and cloning.

Cell sorters now allow the selection of cells and other bodies according to a range of quite diverse criteria. The additional refinement that allows the sorting of individual cells based on these criteria has seen application in many fields of research. Single cells may be sorted for microscopy, for culture and for genetic analysis by way of single cell PCR (polymerase chain reaction). In practical terms, in the setting up of an instrument for single cell sorting, there are additional requirements to ensure that each detected event is indeed a single cell or body, that this cell can be reliably sorted via saline droplet, separate from its fellow travelers, that the aiming of the droplet deflection is sufficiently precise to find the target vessel and that the cell will be undamaged on arrival. Among the diverse reported applications of the technique, two fields which have benefited greatly are lymphocyte development and haemopoiesis. In the former case, the analysis of gene rearrangements in lymphocytes, both in the pre- and post-antigenic phases of development, has been enabled by the combined technologies of single cell sorting and PCR. It is argued that such experiments could not have been done without that partnership. In a similar way, the single cell sorting technique has been found to be the perfect way to demonstrate precursor/progeny relationships between haemopoietic cells and, further, to demonstrate rigorously the effects of particular cytokines on the haemopoietic system.

Cell Separation↗
PubMed1989

[Analysis of cell kinetics after irradiation by flow cytometry (II)--Evaluation of clonogenic potential of G2 block cells after irradiation by cell sorting].

When the tumor cells are irradiated, the percentage of the cells in the G2 + M phase and the duration of the cell cycle arrested increase with the irradiation dose. This post irradiation growth inhibition and changes in the cell kinetics observed within 48 hours of irradiation are closely correlated. Cells in the G2 + M phase are reported to be sensitive to irradiation, but little is known about their clonogenic potential. These cells must be separated for evaluation of their clonogenic potential. In this study, the clonogenic potential of irradiated G2 phase arrested cells was examined by the flow cytometric cell sorting technique. Fluorescent Hoechst staining, which allows determination of DNA content in living cells, was used. The results of DNA assay using Hoechst 33342 agreed well with those using PI staining. The concentration of Hoechst 33342 required for the DNA assay and cell sorting is 5 micrograms at which vital staining is possible with 80% cell viability. Although the clonogenic potential of non-irradiated cells was not different between those in G1 and G2 + M phases, that of the cells arrested in the G2 + M phase after irradiation was lower than that of the cells in the G1 phase. The reduction in the clonogenic potential was more notable in the cells subjected to high dose irradiation. A greater number of cells were arrested for a longer period than the cells irradiated at a lower dose. The clonogenic potential of the cells correlated well with the duration of G2 block.

Cell Separation↗
PubMed1987

Rapid assay for cell age response to radiation by electronic volume flow cell sorting.

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. The sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [3H]thymidine autoradiography of the sorted cell populations, we demonstrate that the resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. The variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. This new method has several advantages, including: no treatment of the cells is required, thus, this method is noncytotoxic; no cell cycle progression is needed to obtain different cell age compartments; the cell population can be held in complete growth medium at any desired temperature during sorting; and a complete radiation age-response assay can be plated in 2 h. The application of this method to problems in radiobiology and chemotherapy is discussed, along with some of the technical limitations.

Animals↗
PubMed1998

Isolation and transplantation of autologous peripheral CD34+ progenitor cells highly purified by magnetic-activated cell sorting.

Peripheral stem cells were mobilized and collected in 26 pediatric patients with malignant diseases. A total of 47 leukaphereses were performed in the 26 patients. The mean number of nucleated cells collected was 4.5 +/- 2.6 x 10(8)/kg and the number of CD34+ progenitors collected was 6.7 +/- 6.8 x 10(6)/kg. CD34-positive selection was performed using a two-step method of magnetic-activated cell sorting (MACS) in 24 patients or a combination of an immunoaffinity column and MACS in two patients. The purity of the positively selected CD34+ progenitors was 98.8 +/- 0.7% and the number of isolated CD34+ cells was 6.5 +/- 5.9 x 10(6)/kg. Thus, the mean recovery of CD34+ cells was 93 +/- 10%. In 22 of the 26 patients, high-dose chemotherapy was performed with subsequent reinfusion of the highly purified CD34+ cells. In all 22 patients, a normal hematopoietic reconstitution was seen with a mean time of 12.4 +/- 2.7 days to reach >0.5 x 10(9)/l neutrophils (range 8-19 days). The time to reach independence from platelet transfusion was 31.6 +/- 17.0 days (range 16-78 days). There were no transplant-related deaths. In summary, we have shown that mobilized peripheral CD34+ progenitors can be highly purified with a good recovery, and that reinfusion of these cells after high-dose chemotherapy results in a rapid, complete and sustained engraftment. We conclude that this method can be used for purging in any CD34-negative malignancies and for autologous T and B cell depletion in the treatment of autoimmune diseases with high-dose immunoablative therapy.

Adolescent↗
PubMed1991

Magnetic-affinity cell sorting of human multidrug-resistant cells.

We have developed a method for isolating multidrug-resistant cells from a heterogeneous cell population using the magnetic-affinity cell-sorting system. Human KB carcinoma cell lines expressing different amounts of P-glycoprotein have been selected by use of the monoclonal antibody MRK-16 coupled to magnetic particles. This specific, rapid, and sensitive method allows the selection of viable and clonable drug-resistant cells from various drug-resistant human and murine cell populations. This method may prove useful in isolating drug-resistant cells from tumors with heterogeneous P-glycoprotein expression for further analysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1983

The characterisation of monoclonal antibodies against haemopoietic cells: comparison of an immunoperoxidase method with fluorescence activated cell sorting.

Nucleated cells from normal human peripheral blood and bone marrow have been analysed with 2 rat monoclonal antibodies of known specificity, one (YAML 555.6.6) directed against the P28,33 complex (anti-HLA-DR), and the other (YAML 501.4.4) directed against leucocyte common antigen (anti-LCA). The patterns of reactivity with an indirect immunoperoxidase method on fixed smeared cells were in close agreement with those obtained with a fluorescence activated cell sorter. A further new monoclonal antibody of unknown antigen specificity (YAML 537.2) reacts with an intracellular antigen present in neutrophils and their precursors from the promyelocyte stage onwards, megakaryocytes, and a proportion of monocytes, but not with eosinophils, nucleated red cells or lymphocytes. This reactivity could not be demonstrated using the fluorescence activated cell sorter. The immunoperoxidase method allows the identification of individual positive and negative cells and therefore provides a method of identifying minor reactive and non-reactive cell populations in a heterogeneous cell sample such as normal bone marrow. Cytoplasmic binding sites can be differentiated from membrane binding.

Animals↗
PubMed2000

Combined use of the green and yellow fluorescent proteins and fluorescence-activated cell sorting to select populations of transiently transfected PC12 cells.

One of the more time-consuming procedures in the study of exogenously expressed proteins in cell lines is the selection of individual transfected clones. In recent years, green fluorescent protein variants with excitation/emission spectra matching the typical flow cytometer configurations have been generated and are in common use. We employed PC12 cells transfected with vectors encoding fluorescent proteins and a fluorescence selection procedure using a fluorescence-activated cell-sorter. In order to select the optimal co-electroporation and sorting conditions, we used the simultaneous detection of two variants of the green fluorescent protein, that possess separable emission peaks when excited at 488 nm. Using these variants and the adequate combination of band-pass filters, we were able to analyze and establish the conditions for identifying and sorting cells transfected with enhanced green fluorescent protein, that simultaneously express another plasmid of interest. Using this procedure, the cells sorted that express both plasmids exceeded 90%. The whole procedure did not alter the physiological responsiveness of the transfected cells to growth factors, and has been successfully applied to the constitutive activation of the mitogen-activated protein kinase pathway, resulting in the spontaneous differentiation of PC12 cells. Also, this procedure has been used with other set of expression vectors encoding proteins that protect PC12 cells from apoptosis caused by different stimuli. The method that we present here provides an easy and fast procedure to obtain a high proportion of positively transfected populations of PC12 cells.

Animals↗
PubMed2000

Arrested maturing multivesicular endosomes observed in a Chinese hamster ovary cell mutant, LEX2, isolated by repeated flow-cytometric cell sorting.

Chinese hamster ovary (CHO) cell mutants defective in the disintegration of endocytosed low-density lipoprotein (LDL) were isolated from mutagenized cells by repeated flow-cytometric cell sorting. After seven rounds of cell sorting, we obtained mutant pools, from which nine mutant clones were established. These mutant strains were all recessive, and were categorized into three complementation groups A, B, and C. The previously established CHO mutant, LEX1 (Lysosome-Endosome X1), fell into the complementation group A. One of the newly isolated mutants, LEX2, fell into the complementation group B, and showed slower degradation of RET-LDL than LEX1 cells. LEX2 showed prominence of well-elaborated multivesicular bodies (MVBs), positive for lysosomal glycoprotein-B/cathepsin D and cation-independent mannose 6-phosphate receptor (CI-MPR), yet negative for transferrin receptor or rab7. Endocytosed intact LDL accumulated in these CI-MPR-positive structures starting at 10-15 minutes of internalization and the accumulation reached completion at 20 minutes. Intermixing of separately internalized fluorescent LDLs between the LEX2 MVBs was slow and saturable at a lower level than observed between late endosomes/lysosomes in wild-type or in LEX1 cells. The receptor recycling pathway to the plasma membrane and the acidification of intracellular compartments were normal in LEX2 cells. These results are consistent with the idea that LEX2 cells are defective in the segregation and sequestration of contents at compartments equivalent to the transport intermediates, previously referred to as endosomal carrier vesicles or maturing MVBs. This MVB stage is likely to be an earlier stage than rab7-positive, lysosome-interacting late endosomes observed in LEX1 cells. Thus, LEX1 and LEX2 mutations could be considered as landmarks for these distinct late endocytic stages, and use of these cells in biochemical and molecular genetic analyses would help to understand the as yet unidentified details of late endocytic pathways including the MVB dynamics.

Animals↗
PubMedDate not supplied

Efficient selection of high-producing subclones during gene amplification of recombinant Chinese hamster ovary cells by flow cytometry and cell sorting.

The screening procedure for high-producing cell lines is extremely time- and labor-intensive and costly, and is at present guided by an empirical approach based on individual experience. Flow cytometry and cell sorting, with its ability to analyze and separate single cells, an ideal method in the selection of such rare cells. The isolation of recombinant cell lines is especially difficult due to repeated gene amplification, which introduces high mutational variation into the population. We have established and evaluated a modification of a previous method that traps secreted product on the surface of the secreting cell, thus allowing direct analysis of single cell specific production rates. This method was used to select for high-producing subclones of a recombinant Chinese hamster ovary (CHO) cell line producing a human antibody against HIV-1 by repeated rounds of gene amplification and cell sorting. This cell line has been amplified in previous investigations, so that the amount of work and testing required by traditional methods can be compared with the protocol described herein. Forty-five 96-well plates were necessary to obtain a high-producing subclone by limited dilution methods, whereas only five plates were required when cell sorting was used. The specific production rate of the best clone obtained by sorting, however, was five times that of the clone obtained by traditional methods. In contrast to the clones obtained by limited dilution, which consisted of several populations of low- and high-producing cells even at high methotrexate concentrations (6.4 microM), the clones isolated by sorting were already homogeneous at 0.8 microM methotrexate.

Animals↗
PubMed1984

Cell sorting and microchemistry of cultured human fibroblasts: applications in genetics and aging research.

Cell sorting is a way to isolate viable subpopulations of cells present in a mixture. The drawback of the isolation method is the shortage of material for subsequent biochemical determinations. We have employed a combination of (ultra-) microchemistry and cell sorting to overcome this problem. The methods enable determinations of protein and several enzyme activities on triton extracts of 5,000-10,000 sorted cells. In addition, using ultramicromethods we could determine enzyme activity in single sorted cells. This combination of methods is used for clinical genetic studies on heterozygote detection in Fabry's disease, an X-linked genetic disease. Moreover, microchemistry is used to study enzyme activities in sorted autofluorescent "aged" fibroblasts.

Aging↗
PubMed2000

A low-voltage droplet charging circuit with simulative cell-sorting function for flow cytometer-cell sorter.

BACKGROUND: Flow cytometer cell sorters have become important tools in many biological laboratories. Commercial electrically-deflected cell sorters that deflect wanted cells in electrically charged droplets need high-voltage amplifiers which are expensive and difficult to obtain. Effort was made to build an alternative droplet charging circuit with low-voltage amplifiers that are much easier to get and have more reasonable price. METHODS: A low-voltage charging circuit was designed. Every time a cell was to be separated, a pair of complementary charging pulses were produced: one was positive and the other was negative with equal amplitude. These were enlarged by two low-voltage charging amplifiers to drive two charging electrodes respectively. RESULTS: Due to the effect of addition, the voltage between the two electrodes was double as high as the output of either amplifier. The result of test experiment proved that the cell sorter with low-voltage amplifiers, which was cheaper and easier to obtain, could separate cells as efficiently as the instrument with high-voltage ones that were more expensive and more difficult to make. In addition, a simulative cell-sorting function was provided. CONCLUSIONS: This low-voltage, easily-built and low-price charging circuit for flow cytometer cell sorter is a good alternative to the commonly used high-voltage one, especially to researcher who hopes to build his own personal instrument.

Animals↗