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The isolation of specific chicken macrochromosomes by zonal centrifugation and flow sorting.

The nine largest macrochromosomes of the chicken, comprising about 80% of the genome, have each been isolated in yields of about 2 x 10(6) chromosomes and approximately 80% purity, except for chromosome 4, which is only about 55% pure because it is heavily contaminated with sex chromosome Z. The chromosomes were first separated roughly according to size by zonal centrifugation on a sucrose gradient. Aliquots of the gradient fractions were then stained with ethidium bromide and analyzed by a flow cytometer-sorter. Electrostatic drop deflection was used to sort the desired chromosomes from chosen zonal gradient fractions. Each fraction was reexamined by flow analysis and by fluorescence microscopy. The DNA from these purified chromosomes has been used for gene mapping by molecular probe analysis.

Animals↗

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↗

Calculation of S-phase numbers of four major cell categories in human bone marrow from DNA-flow cytometry and counterflow centrifugation data.

Fractionation of heterogeneous cell populations into a number of fractions differing in cell size and composition, followed by measurement of DNA profiles and differential morphology of the samples allows the calculation, using a mathematical deconvolution procedure, of the cell proliferation of the individual categories. The method was applied to human bone marrow, fractionated by counterflow centrifugation into 10 to 15 fractions. Calculated percentage of S-phase cells in four major categories, including myeloid, erythroid, monocytic, and lymphoid cells, were in good agreement with data obtained by tritiated thymidine autoradiography.

Bone Marrow↗

Centrifugal separation of cells in sputum specimens from patients with undifferentiated carcinoma.

Ethanol-fixed cells in sputum from patients with undifferentiated carcinoma of the lung were separated in aqueous Ficoll using a discontinuous density gradient centrifugation technique. The selective enrichment of small cell undifferentiated (e.g., oat cell) or large cell undifferentiated carcinoma cells was achieved while removing most of the leukocytes (80-90%) and macrophages (65-75%) from specimen fractions containing the greatest relative frequencies of cancer cells. The maximum purity of small cell carcinoma cells (0.04%) occurs in moderate density (rho = 1.121 g/ml) gradient fractions and results in a 2.4-fold enrichment relative to unprocessed specimens. In contrast, the maximum purity of large cell carcinoma cells (0.22%) is obtained in very high density (rho = 1.172 g/ml) gradient fractions and results in a 1.2-fold enrichment in comparison with unprocessed specimens. Microscopic examination of Papanicolaou-stained specimen fractions reveals that these enrichments were achieved while retaining diagnostically significant cytomorphologic and tinctorial features necessary for cancer screening and diagnosis. Peak purity ranges of undifferentiated cancer cells significantly overlap comparable ranges for material from bronchogenic adenocarcinoma and squamous cell carcinoma.

Carcinoma↗

Separation of rooster spermatogenic nuclei by means of centrifugal elutriation.

By application of minor variations in the usual method of centrifugal elutriation, we have been able to separate a nuclear suspension from rooster testes. We have obtained a population of elongated or late spermatid nuclei (98% purity), a population of round or early spermatid nuclei (90% purity), and, finally, a mixed population of meiotic and pre-meiotic nuclei.

Animals↗

Lymphoreticular cells isolated by centrifugal elutriation from a mammary adenocarcinoma. I. Characterization of an in situ lymphocyte suppressor population by surface markers and functional reactivity.

The procedure of centrifugal elutriation was evaluated as a means of purifying large numbers of in situ lymphocytes from enzymatically disaggregated mouse mammary tumors. The eluate obtained at a flow rate of 3.0 ml/min was optimal for high levels of lymphocyte recovery with low levels of contaminating tumor cells, polymorphonuclear leukocytes, and macrophages. The majority of the tumor infiltrating lymphocytes (90%) expressed the Thy 1.2 antigen, while less than 5% possessed surface immunoglobulin. Further analysis of the T lymphocyte population was accomplished by flow cytometric analysis of in situ lymphocytes stained with fluorescein-conjugated monoclonal anti-Lyt 2 antibodies. The results of such studies reveal an increase in the levels of Lyt 2+ lymphocytes within the in situ population. To determine whether these Lyt 2+ cells were functionally active as suppressor cells, the ISL1 were mixed with spleen cells from tumor bearers and then tested for their ability to respond to mitogen and TAA-induced blast transformation of tumor-bearer spleen cells. Removal of macrophages from ISL by Sephadex G-10 columns did not alter the suppression. Treatment with monoclonal anti-Lyt 1 antibody and complement did not affect the inhibition observed. However, treatment of ISL with anti-Lyt 2+ monoclonal antibody and complement resulted in the elimination of the suppressor cell activity. We concluded that within the tumor-infiltrating lymphoreticular cells there is a population of Thy 1.2+ Lyt 2.2+ lymphocytes responsible for the suppression of mitogen and tumor-antigen-induced blastogenesis.

Adenocarcinoma↗

Comparison of filtration to continuous-flow centrifugation for plasma exchange.

The Asahi Plasmaflo Hollow Nylon Fibre Filtration System (n = 13) was directly compared to the NCI-IBM 2990 Continuous-Flow Blood Fraction Separator (n = 10) for plasma exchange. The systems were equally efficient in achieving plasma separation. There were significant differences favouring filtration for clearance of fibrinogen (P less than 0.05), and the fourth component of complement (P less than 0.01). Greater loss of urea (P less than 0.05) was found after plasma exchange, using the cell separator. The flow characteristics were markedly different. In a standardised 4-L plasma exchange, filtration took place at 35 ml/minute, with a procedure time of 109 +/- 45 minutes in contrast to centrifugation at a plasma flow collection rate of 19 ml/minute, requiring 208 +/- 17 minutes. This time advantage for the former procedure was offset by 195 minutes required to regenerate the hollow nylon fibre unit and a further 90 minutes required for cleaning under strictly controlled aseptic techniques prior to reuse. Each filter was regenerated at least twice and reused without infection, but there was incremental loss of filtration efficiency demonstrated by decreasing clearance of an intravascular marker dye. In two of the 13 procedures using the Plasmaflo system, serious reactions necessitated termination of the procedure; this did not occur using the cell separator. Restriction of the number of times that the filter unit could be regenerated without loss of efficiency, the prolonged time required for regeneration and cleaning, coupled with the need for artificial vascular access to meet high blood flow rates required, limit the usefulness of this technique for plasma exchange.

Blood Chemical Analysis↗

Centrifugal elutriation as a method for isolation of large numbers of functionally intact human peripheral blood monocytes.

Centrifugal elutriation was used further to isolate human peripheral blood monocytes (HPBM) from mononuclear-enriched cells harvested as a secondary component following platelet concentration collection samples. HPBM were recovered in either one or two populations consisting of either total HPBM or small (SM) and large monocytes (LM). The elutriation was carried out at 3,500 +/- 5 rpm for the separation of lymphocytes and HPBM in Ca++- and Mg++-free PBS without EDTA. An average of 5.05 +/- 1.50 X 10(8) HPBM were recovered in the total HPBM with a purity of 95% +/- 3%. The SM and LM were obtained by splitting the total HPBM into two equal populations with an HPBM purity of 92% +/- 3% and 93% +/- 3, respectively, by nonspecific esterase staining. The elutriation media were shown to have no effect on viability by trypan blue exclusion. All three HPBM populations were shown to be histochemically (lack of reactivity to leu-1 and leu-7) and functionally (depletion of NK cell activity) purified from the lymphocyte population. The HPBM populations were enriched in HLA-Dr, OKM-1, OKM-5, MY-8, and leu M-3 monoclonal antibody marker staining. There were no differences in percent positive cells between SM and LM populations for any of the monocyte-specific monoclonal antibodies. All three monocyte populations mediated antibody-dependent cell-mediated cytotoxicity to human red blood cells, with LM mediating more lysis (27.0% +/- 5%) than SM (7% +/- 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

Implementation of a semiclosed large scale counterflow centrifugal elutriation system.

A semiclosed counterflow centrifugal elutriation system that minimizes the risks of contamination and cell loss is presented. A detailed description of the configuration of the system and its sterilization and assembly is provided along with examples of its application to large-scale separation of leukapheresis buffy coat and cadaveric bone marrow. We are currently using this system to deplete lymphocytes from human bone marrow prior to use in allogeneic bone marrow transplantation. The implementation of a semiclosed system increases the safety, ease of operation, and reproducibility of a technique that has the potential for a wide range of clinical applications.

Cell Separation↗

Efficacy of pentastarch in granulocyte collection by centrifugal leukapheresis.

The efficacy of 6% hydroxyethyl starch (hetastarch, HS) in enhancing granulocyte harvest by centrifugal leukapheresis has been described by a simple equation which predicts the granulocyte collection efficiency (GCE) based on an intrinsic donor variable, the erythrocyte sedimentation rate (ESR): GCE(%) = 1.3 ESR (mm/hr) + 45. Ten percent low molecular weight hydroxyethyl starch (pentastarch, PS) has been reported to be as effective as HS with potentially fewer adverse donor reactions (ADR). The derivation of an analogous equation for PS under conditions previously reported for HS may quantify PS efficacy and allow comparison to HS. We prospectively measured the in vitro and the in vivo effects of PS on the donor ESR in 53 granulocyte collections from 44 donors using the model CS-3000 Plus blood cell separator (CS). We then correlated the findings with the GCE of each procedure and derived an equation which expresses GCE in terms of baseline donor ESR. The in vitro addition of PS increased the donor ESR 2.4-fold, but its administration to a donor during a collection procedure did not appreciably change the ESR. Higher baseline donor ESR was more likely to result in more efficient cell collections: GCE (%) = 0.8 ESR (mm/hr) + 20; (r = 0.37). For granulocyte harvests using the CS and PS as the sedimenting agent 1) baseline donor ESR affects granulocyte harvests, but the poor correlation does not allow an accurate prediction of GCE and cell yield from the baseline donor ESR; 2) in comparison with HS (results from a previous study), PS may be less effective in vitro and not effective in vivo in elevating ESR, and may be less effective in enhancing granulocyte harvest; and 3) the parameters (slope, y-intercept, correlation coefficient) which define the linear relationship between baseline donor ESR and GCE may serve collectively as a quantitative measure of the effectiveness of different hydroxyethyl starch agents in enhancing granulocyte harvests. These parameters may be helpful in rapidly assessing the clinical efficacy of new, potentially useful hydroxyethyl starch agents prior to initiating a randomized, controlled clinical trial.

Blood Sedimentation↗

Purification of human granulocytes by centrifugal elutriation and measurement of transmembrane potential.

Viable cell samples containing 93% pure granulocytes were obtained from human blood using the techniques of dextran sedimentation followed by centrifugal elutriation. The resting transmembrane potential (Em) of human granulocytes was estimated using the fluorescent lipophilic cation, Di-S-C3(5), from the null point for potassium--i.e., the external K concentration at which there is no change in Em in response to valinomycin (a K ionophore). The Em of human granulocytes, as calculated from the Nernst potential for K at the null point, is approximately -100 mV. Data indicate that this large transmembrane potential is due in part to the presence of an electrogenic Na-K pump in human granulocytes which is stimulated by external potassium and inhibited by ouabain.

Cell Separation↗

Fertilizing ability of mouse sperm from different epididymal regions and after washing and centrifugation.

The ability of caput, corpus, and cauda epididymal and vasa deferentia mouse sperm to fertilize eggs in vitro was investigated. Cauda epididymal sperm fertilized 45 of 54 ova, whereas caput and corpus sperm fertilized a negligible number of ova, thus indicating their immaturity. Vasa deferentia sperm fertilized only 10 of 52 ova and their reduced fertility may be due to aging in vivo. No difference in preimplantation development of the resulting zygotes with respect to the sperm source was observed. Three centrifugations and resuspensions of cauda epididymal sperm in fresh medium did not affect their fertilizing ability. I conclude that fertilization in vitro can occur after removal of epididymal secretions.

Animals↗

Heterogeneity among alveolar macrophages in humoral and cell-mediated immune responses: separation of functional subpopulations by density gradient centrifugation on Percoll.

Rabbit alveolar macrophages (AM) were separated into four subpopulations by centrifugation on discontinuous density gradients of Percoll. The subpopulations were compared to unseparated AM populations for their ability to provide accessory function to adherent cell-depleted splenocytes for antigen-stimulated lymphoproliferation and for the production of lymphokine. They were also tested for their ability to modulate in vitro plaque-forming (PFC) responses. AM subpopulations that provided accessory function for the production of migration inhibitory factor (MIF)-containing culture supernatants were recovered from the least dense fractions of the Percoll gradients. These cells were cytochemically characterized as mature cells. AM that suppressed the in vitro PFC response and augmented the antigen-stimulated lymphoproliferative response to the greatest degree were recovered from the most dense fractions of the Percoll gradients and were characterized as immature cells. These results suggest that there are distinct subpopulations of AM, the function of which may represent different stages of maturation (or differentiation).

Animals↗

The separation of cytotoxic human peripheral blood monocytes into high and low phagocytic subsets by centrifugal elutriation.

Mononuclear cells (4 X 10(8) in a 5-ml volume) were loaded onto a Beckman JE-6B elutriator rotor spinning at 2,000 +/- 10 rpm with a flow rate of 10.0 ml/min. After lymphocytes were removed at flow rates between 10 and 11 ml/min with 500 ml of buffer, the flow rate was increased by 0.5 ml/min/fraction to collect 100-ml fractions. Highly enriched monocytes as judged by nonspecific esterase staining and morphology (70-95%) were found in each of eight fractions collected with flow rates between 11.5 to 15.0 ml/min. When stimulated with phorbol myristic acetate, these fractions mediated equivalent levels of cytotoxicity against 51Cr-labeled Chang liver cell line. Similarly, each monocyte-containing fraction was found to mediate the same level of cytotoxicity against antibody-sensitized 51Cr-labeled Chang liver cells. In contrast, cytotoxicity against the natural killer cell-sensitive K-562 cell line was found in only those fractions that contained a high percentage of lymphocytes. The fractions that were enriched in monocytes were found to differ in their ability to ingest latex. Those monocyte fractions that were collected between 11.0-12.0 ml/min consisted primarily of low numbers of latex-ingesting monocytes (less than 30%). Those monocyte fractions that were collected between 12.5 and 15.0 ml/min consisted primarily of latex-ingesting monocytes (50-70%). These data show that cytotoxic monocytes can be separated by centrifugal elutriation into at least two subsets that can be distinguished by their phagocytic activity.

Animals↗

Volume dependent polymorphonuclear leukocytes fractions isolated by counterflow centrifugal elutriation: PMN volume does not correlate with PMN age.

We have previously reported that human peripheral blood polymorphonuclear leukocytes can be separated into at least six volume dependent fractions using counterflow centrifugal elutriation (CCE). Larger volume was shown to correlate with increased superoxide release with either the chemotactic peptide F-met-leu-phe (FMLP) or the tumor promotor phorbol myristate acetate (PMA). To help explain these findings we have examined volume dependent PMN fractions (VDPF) for PMN functions felt to be correlated to PMN age, ie, alkaline phosphatase activity, phagocytosis of opsonized particles, adherence to plastic, and directed movement to FMLP and zymosan activated serum. PMN alkaline phosphatase activity was found to be low in the smallest PMNs, increasing in the PMNs of intermediate size, and then decreasing again in the largest PMN functions. A similar relationship was noted for PMN phagocytic activity. No significant difference among VDPF in adherence to plastic or chemotaxis was found. Furthermore, no difference among VDPF was seen for receptor number or binding affinity of the ligands FMLP or phorbol dibutyrate. Total cellular activity of the NADPH dependent oxidase was, however, 200% greater in the largest compared to the smallest VDPF. Because of the lack of consistent correlation between "age" related PMN function and PMN volume, it is unlikely that PMN volume represents a manifestation of PMN age. It is likely that the increased oxidase activity seen among larger VDPF accounts for the more rapid oxidative burst previously noted.

Adult↗

Morphologic and functional heterogeneity among rat alveolar macrophage fractions isolated by centrifugation on density gradients.

Lavaged rat bronchoalveolar cells were separated into four different density fractions (I-IV) by centrifugation through a discontinuous Percoll gradient. Maximum cell size was found in the lowest density fraction (I) and minimal cell size in the highest density fraction (IV), showing an inverse correlation with cell density. These fractions contained alveolar macrophages (AM) in the proportion of 97% or more by morphologic criteria, while phagocytic AM was approximately 80% in each fraction. Although the proportion of Ia antigen-positive AM was low in each fraction, it was elevated after incubation with supernatants from concanavalin A (Con A) stimulated spleen cell cultures, with greater Ia expression in higher density fractions. Differences in these fractions were also noted in several functions, including Fc receptor activity, chemotactic migration, tumoricidal activity, and interleukin 1 (IL-1) production, all of which were greater in higher density fractions (III and IV). Con A-induced T cell proliferation was, however, suppressed by these higher density fractions, whereas only an intermediate density fraction (II) enhanced T cell responses. These results indicate morphologic and functional heterogeneity among rat AM.

Animals↗

Transglutaminase levels and immunologic functions of BCG-elicited mouse peritoneal macrophages isolated by centrifugal elutriation.

BCG-elicited mouse peritoneal macrophages were separated into three subpopulations by counterflow centrifugal elutriation. The three subpopulations were characterized on the basis of the level of a protein cross-linking enzyme, tissue transglutaminase. Subpopulation-3 consisted of large cells (greater than 95% esterase positive and greater than 90% viable) and had at least a fivefold higher transglutaminase activity (35 +/- 6 nmol/hr/mg) as compared to macrophages in subpopulation-1 (6 +/- 2 nmol/hr/mg) and at least a threefold higher enzyme activity as compared to subpopulation-2 (11 +/- 2 nmol/hr/mg). Subpopulation-3 also showed sevenfold higher phagocytosis of IgG-coated sheep red blood cells. The three subpopulations showed no difference in their ability to kill Listeria monocytogenes as determined by [3H]-thymidine release. Subpopulations-2 and -3 caused 90% inhibition of murine adenocarcinoma (EMT-6) tumor cell growth in the presence or absence of lipopolysaccharide. Subpopulation-1 had a poor ability to inhibit EMT-6 cell growth (29 +/- 12%). However, in the presence of lipopolysaccharide, this activity increased by at least threefold (92 +/- 7%). The three subpopulations showed no significant difference in their cytolytic activity against murine mastocytoma (P815) target cells in the presence or absence of lipopolysaccharide. These results suggest that tissue transglutaminase may have no significant role in bactericidal, tumoricidal, or tumoristatic function of macrophages; however, it might have some role in promoting the Fc-receptor-mediated phagocytic function of the macrophages.

Animals↗