Cell Dynamics in Phytohemagglutinin (PHA) Stimulated Guinea-Pig Lymph Node Lymphocyte Cultures

Eurenius, Karl; McIntyre, O. Ross

doi:10.1159/000230802

Cited by 7 publications

(9 citation statements)

References 21 publications

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“…Figure 1 illustrates the growth rate as measured by tritiated thymi dine incorporation in PHA stimulated cultures as compared with non stimulated cultures. The rate of DNA synthesis in these large cultures containing a total of 50 to 60 million cells is comparable to that found in previous cultures containing a total of 2 million cells [5]. Measure ments of culture total cell mass ( fig.…”

Section: Methodssupporting

confidence: 83%

“…It is this latter binding which relates directly to glucocorticoid activity on the cell [2], In previous studies [3,4] we examined the total steroid binding to various populations of lymphocytes before and shortly after exposure to the mitogen phytohemagglutinin (PHA). To examine steroid bind ing in cell cultures exposed to PHA for periods of time required for cell transformation, methods of cell counting and determination of culture mass were first evaluated, using guinea-pig lymph node lymphocytes as a model [5]. These studies were necessary to determine the packed cell volume of lymphocytes after the marked cellular changes occurring with transformation in cultures containing suffi cient cells to determine steroid binding.…”

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confidence: 99%

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The Mechanism of Glucocorticoid Action on the Phytohemagglutinin-Stimulated Lymphocyte

Eurenius¹,

McIntyre²

1970

Int Arch Allergy Immunol

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Section: Methodssupporting

confidence: 83%

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confidence: 99%

The Mechanism of Glucocorticoid Action on the Phytohemagglutinin-Stimulated Lymphocyte

Eurenius¹,

McIntyre²

1970

Int Arch Allergy Immunol

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“…Cell size distribution and mean cell volume distribution were accomplished, with a dual threshold electronic particle counter (Coulter Electronics Model B), in 20 //3 * increments from 100 to 500//3 [10]. Samples were diluted 1:50 in normal saline, and corrections made for background and debris.…”

Section: Methodsmentioning

confidence: 99%

The Phytohemagglutinin (PHA) Response in the Thermally Injured Rat

Eurenius¹,

Mortensen²

1971

Int Arch Allergy Immunol

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“…Our interest in quantitation of cell death was stimulated by the observations made by ourselves and others (2)(3)(4)(5)(6)(7)(8) that in phytohemagglutinin (PHA)-stimulated lymphocyte cultures, at widely used cell concentrations (2 X 106 lymphocytes per ml), an increase in cell number or DNA content is most often not demonstrable, in spite of considerable thymidine (Td) incorporation and mitotic activity, raising the question of the fate of newly synthesized DNA. It has been demonstrated that after mitogen stimulation of lymphocytes a major portion of the newly synthesized DNA is eventually found in the culture medium (5,9,10).…”

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confidence: 99%

“…This DNA contains specific, apparently amplified sequences that may be present in 3-to 4-fold greater amounts in phytohemagglutinin (PHA)-stimulated than in noncycling human lymphocytes (11,12). Because cell death was not accurately quantified in these studies (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), it was of interest to evaluate its overall importance in lymphocyte cultures and to determine the fate of DNA in dead cells. Abbreviations …”

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Kinetics of cell death and disintegration in human lymphocyte cultures.

Bernheim

Mendelsohn²,

Kelley

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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In order to quantitate lymphocyte proliferative responses, we explored the role of cell death in the kinetics of phytohemagglutinin-stimulated cultures. Unless the disintegration time (tDIS) of nonviable lymphocytes in culture is known, the rate&'Gcell death cannot be calculated. To obtain tDIS, we determined the time interval between total and viable cell population decay after various killing events. Two subpopulations of Iymphocytes were observed, the major (80%) with a mean (+ SEM) tDIS of 16 + 2 hr and the minor (20%) with a tDIS of 45 7 hr. Kinetic balance sheets were constructed predicting total cultuire DNA content (cells plus medium), as calculated both from proliferation rates and from observed death and disintegration rates. In an experiment characterized by extensive cell death, the two tallies were well-matched when the above data were utilized. The large discrepancy between predicted and observed DNA contents of the medium indicates that the DNA of disintegrated lymphocytes is extensively degraded. We conclude that cell death explains proliferation deficits in stimulated lymphocyte cultures. Our MATERIALS AND METHODSHuman lymphocytes from normal volunteer donors were purified (>95%) and cultured according to our published methods (13,14). Weakly agglutinating, purified PHA (Wellcome Reagents Ltd., Beckenham, England) was added to bulk cultures containing 2 to 33 X 105 cells per ml to a final concentration of 3 ,tg/ml, which gave optimal stimulation. Cultures were pulsed with [3H]Td, 2 ,uCi/ml (6 Ci/mmol) to assess incorporation into acid-precipitable material and autoradiographic labeling indices (15). The DNA contents of cell pellets and culture medium were assayed fluorometrically with diaminobenzoic acid after acid precipitation and extractions with alcohol and ether (16). Enumeration of Living and Dead Cells. The total cell number was determined in quadruplicate in a hemacytometer. The percentage of cells with a blue nucleus after incubation in 0.1% trypan blue for 5 min at room temperature was determined as the mean of quadruplicate counts of 100 cells each.Only those stained lymphocytes that had a morphology sufficiently unaltered for them to be included in the total cell count were scored as dead, i.e., blue-stained debris was ignored. Alternatively, viable (Pronase resistant) and dead cell nuclei were counted with a Coulter model D RESULTSMeasurement of sensitivity to killing events and tDIS by population decay In the population decay analysis (Fig. 1), irradiated cells that are dead at time t1 will have disintegrated at some later time t2, when the total cell number (alive plus dead) will-represent only the cells that were viable at time t1. The interval t2 -t 2536

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Cell Dynamics in Phytohemagglutinin (PHA) Stimulated Guinea-Pig Lymph Node Lymphocyte Cultures

Cited by 7 publications

References 21 publications

The Mechanism of Glucocorticoid Action on the Phytohemagglutinin-Stimulated Lymphocyte

The Mechanism of Glucocorticoid Action on the Phytohemagglutinin-Stimulated Lymphocyte

The Phytohemagglutinin (PHA) Response in the Thermally Injured Rat

Kinetics of cell death and disintegration in human lymphocyte cultures.

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