Purification of human T and B cells by a discontinuous density gradient of percoll

Gutiérrez, Carmen; Bernabé, Rosa R.; Vega, Jesús; Kreisler, M.

doi:10.1016/0022-1759(79)90125-x

Cited by 121 publications

(28 citation statements)

References 9 publications

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“…The density range in which human peripheral-blood cells band in our gradients is slightly lower than that obtained by Pertoft et al (1979), Gutierrez et al (1979 and Ulmer & Flad (1979), who used different centrifugation and Percolldilution protocols. We do not believe that these minor differences in density are due to the labelling procedure, because very similar density distributions were obtained with unlabelled PBC.…”

Section: Stability Of Buoyant Density Of Tumour Celllinesfrom Culturecontrasting

confidence: 68%

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A rapid method for the isolation of metastasizing tumour cells from internal organs with the help of isopycnic density-gradient centrifugation in Percoll

Bosslet¹,

Ruffmann²,

Altevogt³

et al. 1981

Br J Cancer

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Metastasizing tumour cells from a DBA/2 mouse T-cell lymphoma could be separated from the invaded tissue by isopycnic centrifugation in continuous Percoll density gradients. The metastasizing tumour cells from spleen, liver and lung, derived from a cloned lymphoma-cell line, showed a buoyant density in Percoll of 1.060 +/- 0.010. They could be separated from the host tissue, which had a higher buoyant density in the case of the spleen cells or a lower density in the case of the dead liver or lung tissue. The separated tumour cells as removed from the gradients were viable, and could be analysed by in vitro and in vivo assays. The separation procedure did not affect the expression by the tumour cells of TATAs and H-2 antigens. Furthermore, the method seemed to be applicable to the separation of human tumour cells from mononuclear cells prepared from blood samples of tumour patients by Ficoll centrifugation.

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Section: Stability Of Buoyant Density Of Tumour Celllinesfrom Culturecontrasting

confidence: 68%

“…Such density differences could be used to separate cell subpopulations (Gutierrez et al, 1979;Kurnick et al, 1979) and subcellular components in animals and man (Jenkins et al, 1979 (Bosslet et al, 1979).…”

mentioning

confidence: 99%

A rapid method for the isolation of metastasizing tumour cells from internal organs with the help of isopycnic density-gradient centrifugation in Percoll

Bosslet¹,

Ruffmann²,

Altevogt³

et al. 1981

Br J Cancer

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“…suspensions were obtained by either anti-Thy 1,2 antibodies and complement ( 1 ), or Percoll density gradients (4).…”

mentioning

confidence: 99%

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes.

Pozzan¹,

Arslan²,

Tsien³

et al. 1982

The Journal of Cell Biology

208

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This paper examines, in mouse spleen lymphocytes, the effect of anti-immunoglobulin (anti-Is) on the cytoplasmic free calcium concentration, Capping of surface Ig represents a dramatic reorganization of membrane components apparently involving cytoskeletal elements and requiring metabolic energy (2, 12). Formation of caps is followed by contractile activity and cell motility. Rather little is known of how ligand binding stimulates these processes although changes in ion flux and potential difference across the plasma membrane play no essential part (7). Ca 2+ ions are well known to trigger muscle contraction and are implicated in the regulation of various forms of nonmuscle motility, so it has been attractive to suppose that a rise in cytoplasmic free Ca z+ mediates the B cell response to anti-Ig (2). The evidence hitherto available is entirely indirect. It is generally agreed that external Ca z÷ is not needed. The ionophore A23187, which in other cell types raises cytoplasmic free [Ca 2+] and stimulates Ca2+-activated processes, actually inhibits capping in B lymphoeytes. However this inhibitory effect, like those of local anesthetics (5) and cis-unsaturated fatty acids (3), is at least in part explainable (10) by depletion of cellular ATP. Recently anti-Ig has been found to cause a rapid loss of 45Ca from mouse spleen cells (l). This efflux was interpreted as mobilization of Ca 2+ from internal stores causing a rise in the level of free cytoplasmic Ca 2+ and subsequent extrusion of the mobilized ions. There was no evidence that the Ca 2+ movements caused capping but the correlations between the two were felt to suggest "that Ca 2+ may have a physiological role in contractile dependent capping" (2).In the present paper the intracellular Ca 2+ indicator, quin2 (13, 14), has been used to assess more directly the importance of Ca 2+ by monitoring and manipulating the cytoplasmic free Ca 2+ concentration, [Ca2+]i, in mouse spleen lymphocytes. The changes in [Ca2+]i brought about by anti-Ig on cap formation are analyzed and the effects of preventing such changes are examined. MATERIALS AND METHODSBALB/c mouse spleen lymphocytes and thymocytes were prepared as previously described (6-10) in HEPES-buffered culture medium, either H-M EM or RPM 1-1640. Viability, assessed by eosin exclusion, exceeded 95%. B-cell enriched

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“…Monocyte suspensions contained 70-75% lymphocytes and were further purified using the rate zonal centrifugation method [24] modified by Dr. A.C. Bloem (Department of Clinical Immunology, University Hospital Utrecht, The Netherlands). 1-10 7 monocytes were centrifuged for 10 min at 160 x g, and the supernatant solution was discarded and resuspended in 1 ml 90% (v/v) Percoll (9 parts of Percoll mixed with 1 part of 10 times concentrated phosphate-buffered saline).…”

Section: Methodsmentioning

confidence: 99%

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function

Nijkamp¹,

Henricks²,

Tol³

et al. 1984

Biochimica et Biophysica Acta (BBA) - General Subjects

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Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 #M arachidonic acid, neutrophils (5.106 cells/ml) produced superoxide (53 + 8 nmol/5.106 cells per 15 rain), generated chemiluminescence (1211 100 + 157000 cpm) and consumed oxygen (20 + 1 nmol/106 cells per 5 rain). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 /xM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 + 8 nmol superoxide/5 • 106 cells per 15 min and generated 27000 + 15000 cpm chemiluminescence when stimulated with 80 /~M arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells.

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Purification of human T and B cells by a discontinuous density gradient of percoll

Cited by 121 publications

References 9 publications

A rapid method for the isolation of metastasizing tumour cells from internal organs with the help of isopycnic density-gradient centrifugation in Percoll

A rapid method for the isolation of metastasizing tumour cells from internal organs with the help of isopycnic density-gradient centrifugation in Percoll

Anti-immunoglobulin, cytoplasmic free calcium, and capping in B lymphocytes.

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function

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