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PubMed1984

Enumeration of human lymphocyte subsets by monoclonal antibodies and flow cytometry: a comparative study using whole blood or mononuclear cells separated by density gradient centrifugation.

Enumeration of lymphocyte subpopulations by combined use of flow cytometry and monoclonal antibodies is influenced by the separation method used. T or B lymphocyte antigen frequencies do not differ in samples of whole blood or after separation on Ficoll-Paque or Percoll, but there is a significant increase of Leu+ Leu11+ lymphocytes showing strong natural killer activity at the sample-separation medium interface. At the bottom of the tubes a selective loss of OKT+, Leu7+, Leu11- cells is found; at this level cytotoxicity is very low. Our data suggest that Leu7+ cells could be subdivided into 2 subpopulations differing in reactivity with the monoclonal antibody to Leu11, natural killer activity and density. Differences observed in the percentages of Leu7+, Leu11+ lymphocytes between whole blood and separated cells could be due to an enrichment produced by centrifugation or to the presence in whole blood of a factor interfering with antibody binding.

Antibodies, Monoclonal↗
PubMed1984

Methods for using centrifugal elutriation to separate malignant and lymphoid cell populations.

Malignant cells and non-neoplastic host cells were isolated from in vivo growing solid tumors of 2 BALB/c transplantable tumors: EMT6/Ro, a mammary sarcoma, and Line 1, an alveolar cell carcinoma. The technique of centrifugal elutriation was used to separate these 2 major types of cells present within solid tumors. In addition it was found that 2 successive passages through the elutriator of the cells from the tumors resulted in a better separation of the host cells into lymphocyte and macrophage populations than was possible by an optimally designed single passage. Thus this technique can provide sufficient host cells, essentially devoid of malignant cells, for further functional analysis.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1984

Collection of large quantities of mononuclear cells from individual donors by continuous flow centrifugation leukapheresis.

Continuous flow centrifugation leukapheresis allows the continuous removal of leukocytes from the peripheral blood of individual donors. With the IBM 2997 cell separator, leukapheresis of granulocytes has been well established. On the other hand, no standard procedure for the collection of lymphocytes and/or monocytes has yet been described. Here, we investigated the conditions necessary for collection of large amounts of mononuclear cells with highest possible purity. The average yield from more than 100 healthy donors was 8 X 10(9) leukocytes per donor containing 80-100% mononuclear cells (MNC). Of the MNC, 10-55% were monocytes and 45-90% lymphocytes, depending on the donor. The rotor speed for maximal MNC collection was between 800 and 1000 rpm. Highest yields of monocytes were obtained at 900 rpm. No sedimenting agents were added to the blood. With more than 150 donors no rebound leukocytosis or lasting depression of blood cell counts was observed after leukapheresis. One patient with the Sézary syndrome who was subjected to leukapheresis for 26 months at 3-6 week intervals showed no pathological changes in his blood cell count to date. The disease was stabilized and no abnormalities in resistance to infection became apparent.

Adult↗
PubMed1984

Isolation of human splenic macrophages and lymphocytes by countercurrent centrifugal elutriation.

Certain tissues, such as the spleen, are rich sources of mononuclear phagocytes (MP); however, separating the phagocytes from tissues and removing the contaminating cells have been difficult. We report here a method for the extraction and purification of human splenic MP that employs gentle homogenization of splenic fragments with a Tenbroeck tissue homogenizer, controlled digestion with purified collagenase to free MP from splenic stroma, incubation with DNase to dissociate cell clumps and purification by countercurrent centrifugal elutriation (CCE). With homogenization and enzymatic digestion most of the splenic nonspecific-esterase-positive cells are freed into suspension as determined by morphometric analysis of 2 micron sections from plastic embedded spleen stained for alpha-naphthyl butyrate esterase (ANB). Overall cell recovery after homogenization and enzyme treatment is 56 +/- 7%; no selective cell loss occurs as determined by differential cell counts at each purification step. CCE of up to 3 X 10(9) treated spleen cells results in recovery of 63 +/- 6% of the elutriated cells and separates nearly 50% of the recovered MP into enriched fractions. These MP are morphologically intact as determined by light and electron microscopy and are actively phagocytic. Highly purified (96%) autologous splenic lymphocytes are a useful by-product of this separation technique.

Cell Count↗
PubMed1984

Lymphocyte isolation from human spleen by counterflow centrifugation employing two different flow chambers on line.

Studies on splenic lymphocytes have hitherto been performed on single cell suspensions depleted of phagocytic cells by adherence to plastic or incubation with carbonyl iron. These techniques have the disadvantages of selective cell loss, suboptimal cell purification and cell activation. This paper describes purification of splenic lymphocytes by the use of counterflow centrifugation (CFC). The method was adapted to overcome pelleting of cells in the separation chamber to form a plug at the inlet and impede adequate flow. By combining 2 different separation chambers on line in 1 rotor this problem was overcome. Of all lymphocytes recovered after CFC 88.8 +/- 1.4% were collected in 2 pooled fractions with a purity of greater than or equal to 98% and a cell viability of 95%. After CFC, 80.8 +/- 12.1% of the viable cells loaded were recovered.

Blood Platelets↗
PubMed1986

Single step enrichment of human peripheral blood basophils by Ficoll-Paque centrifugation.

A simple, rapid and economical method for the enrichment of basophils from peripheral blood with high recovery is described. Peripheral blood was layered over a Ficoll-Paque cushion and centrifuged. The Ficoll-Paque layer was aspirated taking care not to disturb the interphase and the buffy coat. The Ficoll-Paque layer contained most of the basophils. A recovery of 82% (65-95%) and a 23.5-fold enrichment of basophils with their morphology and biological activity preserved was achieved.

Antibodies, Anti-Idiotypic↗
PubMed1986

Large scale separation of human bone marrow by counterflow centrifugation elutriation.

Counterflow centrifugation elutriation (CCE) has been used to separate moderate quantities of bone marrow (BM) into distinct cell populations for further in vitro investigation. Recently, this technique was employed to reduce the incidence of graft-versus-host disease (GVHD) in human allogeneic bone marrow transplantation (BMT) by removing the majority of mature lymphocytes from the graft. Unfortunately, current methods employing the small (J6-B) elutriator rotor require time consuming preseparation steps and multiple runs. We report our experience with a fixed rotor speed, two-flow rate elutriation procedure using the new Beckman JE-10X rotor system which can separate more than 10(10) nucleated BM cells in 30 min. Three fractions were obtained which differed in size, morphology, and immunologic capacity. The large cell fraction is suitable for allogeneic BMT since it is radically depleted of mature T lymphocytes and retains high clonogeneic capacity.

Bone Marrow Cells↗
PubMed1988

Rapid purification of human peripheral blood monocytes by centrifugation through Ficoll-Hypaque and Sepracell-MN.

We have developed a rapid and simple method for isolating human peripheral blood monocytes in suspension. The procedure combines two separation media and involves isolation of the mononuclear cells by centrifugation through Ficoll-Hypaque followed by purification of the monocytes using Sepracell-MN, a colloidal silica-based medium. The final cell population contained approximately 90% monocytes with good functional ability. The contaminating cells were lymphocytes. Viability was always greater than or equal to 99% with 90% recovery of the monocytes from the mononuclear cells.

Cell Separation↗
PubMed1989

A single step centrifugation method for the enrichment of veiled cells from canine afferent lymph.

Veiled cells (VC) present in the afferent lymph of dogs with chronic lymphoedema could be enriched from 6% to about 50% VC by density gradient centrifugation on 15% metrizamide or discontinuous Percoll gradients. The recovery of VC was about 40% from 0.22 +/- 0.07 X 10(6) VC/ml of lymph. The cells were strongly Ia positive and had cytoplasmic S 100 protein. They were also strongly ATP-ase positive and showed heterogeneity in acid phosphatase, peroxidase and non-specific esterase activity. Low density VC from canine afferent lymph were able to stimulate both blood and lymphatic lymphocytes in autologous mixed leukocyte reaction when present at concentration as low as 5% of cultured cells.

Acid Phosphatase↗
PubMed1993

Optimizing follicular dendritic cell isolation by discontinuous gradient centrifugation and use of the magnetic cell sorter (MACS).

Follicular dendritic cells (FDC) contribute minimally to the total cell population of lymphatic tissue. In order to obtain higher numbers of viable FDC with only a small fraction of contaminating cells the following procedure was developed. Subsequent to the usual mechanical and enzymatical digestion of human tonsils, single cells were layered on top of a discontinuous bovine albumin gradient and centrifuged at 8500 x g. The suspension collected from the 1.052-1.030 interphase contained an average of 10.5% FDC. Next, the preparation was subjected to a new step involving separation of FDC previously treated with biotin-labelled KiM4 monoclonal antibody, raised against FDC, and attached via biotin-streptavidin bonding to streptavidin-conjugated paramagnetic beads. Purification on a magnetic cell sorter (MACS) yielded 3.3-10.1 x 10(6) cells with an average FDC content of 78.4%. The viability and morphology of the resulting FDC population was examined using trypan blue staining or electron microscopy. This technique will permit in vitro studies and long term cultures with FDC isolated from human lymphatic tissue.

Antibodies, Monoclonal↗
PubMed1993

Purification of murine Kupffer cells by centrifugal elutriation.

Procedures to reproducibly obtain pure preparations of murine Kupffer cells are described. Pure Kupffer cell preparations obtained following collagenase digestion, metrizamide separation and centrifugal elutriation remained viable and maintained their phagocytic functions for at least 4-5 days in vitro. Furthermore, we determined the feasibility of extracting RNA from Kupffer cells obtained immediately following elutriation and after 2 and 4 days of culture in vitro. These RNA extracts were used to determine the level of cytokine gene expression in Kupffer cells.

Animals↗
PubMed1995

Isolation of human blood dendritic cells by discontinuous Nycodenz gradient centrifugation.

The most potent antigen presenting cell present in peripheral blood, lymphoid and non-lymphoid tissue is the dendritic cell (DC). The study of human DC has been restricted by their low frequency in the tissues and the lack of a truly DC specific surface marker to assist in identification and isolation. Standard techniques for the isolation of blood DC generally employ a period of in vitro culture followed by flotation on dense albumin gradients, or more recently, discontinuous gradients of metrizamide. Dense albumin gradients are time consuming to prepare, giving low and variable yields of DC. Metrizamide is more convenient, although exposure of monocytes to metrizamide can decrease the expression of CD14 and alter the accessory cell properties of antigen presenting cells. Here we demonstrate that Nycodenz gradient centrifugation of 16 h cultured, T lymphocyte depleted, peripheral blood mononuclear cells (PBMC) reliably yields a population of low density cells that is highly enriched for DC. Most B and residual T lymphocytes are depleted and NK cell numbers are reduced two-fold from the interface cell population. The high density pellet fraction exhibits very little allostimulatory activity, indicating that few DC pass into the pellet. The low density fraction contains a significant population (20 +/- 5 (SD)%, n = 8) of cells which fail to stain for the lineage markers CD3, CD11b, CD14, CD16, CD19 and CD57. Nycodenz exhibits low toxicity, does not alter the allostimulatory activity of antigen presenting cells, and is therefore ideal for the isolation of cultured DC.

Cell Separation↗
PubMed1985

Evaluation of an improved centrifugal casting machine.

A Type III gold alloy, a silver-palladium alloy, and a base metal alloy were cast in two different centrifugal casting machines. With the number of complete cast mesh squares as an indicator of castability, the Airspin casting machine produced superior castings with all three alloys. The base metal alloy produced the greatest number of complete squares with both casting machines.

Centrifugation↗
PubMed1984

Validation and use of centrifugal ultrafiltration-dialysis in the measurement of percent free oestradiol in serum.

The validity of the centrifugal ultrafiltration-dialysis method in the measurement of the percentage of oestradiol and testosterone which is not bound to protein (free) in human serum was assessed. In an initial validation, the values of % free steroid obtained for oestradiol and testosterone in protein-free ultrafiltrates of human serum were higher than expected. Filter paper discs used in the system were shown to adsorb [3H]oestradiol from protein-free solution. Their replacement with glass-fibre discs, which did not adsorb oestradiol, decreased but did not eliminate the discrepancy between observed and expected results for % free oestradiol in ultrafiltrates. The use of polyethylene rather than polypropylene vials then reduced the discrepancy to insignificant levels. The estimates for % free oestradiol in serum were reduced by these modifications.

Centrifugation, Density Gradient↗
PubMed1987

Effects of centrifugal rotation on analgesia and conditioned flavor aversions.

In the first experiment, 48 female Wistar rats were water deprived and given three conditioning days with saccharin-flavored water (1.5 g/liter) followed by 0, 5, 10 or 15 min of centrifugal rotation (150 rpm). Analgesia was measured by the tail flick test immediately after rotation. Over conditioning days, taste aversions developed. In general, taste aversion strength increased with duration of rotation. Analgesia also was in proportion to duration of rotation; however, over days tolerance developed in all rotated groups. In the second experiment 12 female Wistar rats were water deprived and given naloxone (20 mg/kg) or saline prior to 15 min of rotation. Rotation-induced analgesia was not affected by naloxone. It was concluded that somatic and gastrointestinal reactions to rotation are not served by a single mechanism.

Animals↗
PubMed1982

The purification of gametocytes of Plasmodium falciparum and P. yoelii nigeriensis by colloidal silica (Percoll) gradient centrifugation.

Percoll gradient centrifugation has been applied to Plasmodium falciparum- and P. yoelii-infected blood to allow rapid concentration of gametocytes. In the case of cultured P. falciparum, stage IV and V gametocytes were separated from immature stage II and III forms and concentrated from initial gametocytaemias of 1 to 3% to an average of greater than 80% and a maximum of 100% purity in a fraction which collected at the 1.064/1.083 g/ml interface; the rounded gametocytes of P. yoelii-infected mouse blood were also concentrated in this fraction. A much larger enrichment in microgametocyte concentration (average of 160-fold) was recorded in a fraction that collected at the 1.047/1.064 g/ml interface. The large increases in gametocyte concentration were paralleled by similarly large increases in exflagellation activity, thus confirming the viability of both preparations.

Centrifugation, Density Gradient↗
PubMed1987

Plasmodium vivax: isolation of mature asexual stages and gametocytes from infected human blood by colloidal silica (Percoll) gradient centrifugation.

The densities of human erythrocytes infected with P. vivax obtained from infected patients were determined by isopycnic centrifugation in continuous gradients of Percoll. The approximate densities of erythrocytes infected with rings were 1.086 to 1.1, trophozoites (amoeboid stages) 1.053 to 1.086, schizonts and gametocytes 1.053 to 1.056 and of the other cellular elements of blood, uninfected erythrocytes, 1.086 to 1.1, polymorphonuclear leucocytes 1.073 to 1.086 and mononuclear cells 1.062 to 1.073 g/ml. Based on these values, a one-step gradient of 47% Percoll was devised to separate erythrocytes infected with the more mature stages (trophozoites, schizonts and gametocytes) from uninfected erythrocytes. By this method it is possible to obtain parasitaemias of 88% to 98% from blood with starting parasitaemias of less than 0.7%. This method is therefore being routinely used for immunological, biochemical and molecular biological studies on P. vivax.

Animals↗
PubMedDate not supplied

Isolation of spotted fever group rickettsias from triturated ticks using a modification of the centrifugation-shell vial technique.

A modification of the centrifugation-shell vial technique was used to isolate spotted fever group (SFG) rickettsias from triturated ticks which had previously been stored at -80 degrees C for up to 7 months. SFG rickettsias were successfully isolated in Vero cells from all 7 haemolymph positive ticks (Amblyomma hebraeum) used in the experiment. Attempts were also made to use these tick triturates to establish SFG rickettsial infections in tissue culture. Vero cells were used for 5 of the tick triturates and human embryonic lung cells (HEL) for the remaining 2. Infections were successfully established in 4 of the 5 Vero cell cultures, but in none of the HEL fibroblast cultures.

Animals↗