Evaluation of the mtt bioassay to determine in vitro proliferation of fresh and frozen bovine lymphocytes

Carlos Ramón Bautista Garfias; Elizabeth Acosta García; Icela Ivonne Toledo García

2000, Number 2

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Vet Mex 2000; 31 (2)

Evaluation of the mtt bioassay to determine in vitro proliferation of fresh and frozen bovine lymphocytes

Bautista GCR, Acosta GE, Toledo GII

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ABSTRACT

Four experiments were carried out to evaluate the in vitro proliferation (expressed as stimulation index, SI) of fresh and/or frozen bovine lymphocytes to Concanavaline A (Con A) and/or Anaplasma marginale antigen using the MTT bioassay. In the first experiment, the SI of lymphocytes from three adult bovines in response to Con A was determined during six consecutive weeks using the MTT assay which showed reproducible results during this time. The SI oscillated between 1.7 and 3.8. In the second one, the lymphocytes from two adult bovines were frozen at -70ºC during six weeks, and their viability was evaluated weekly. Mean viability changed from 99.5% (week 0) to 89% in week five, and dropped to 59% in the sixth one. In the third experiment, the SI of fresh and frozen (three weeks) lymphocytes from three adult bovines in response to Con A was evaluated using the MTT bioassay; the mean SI was of 2 and of 1.5 for fresh and frozen lymphocytes, respectively. In the fourth experiment, the proliferation of lymphocytes from one adult animal recovered from A. marginale infection, and those from one non infected adult bovine were tested against an A. marginale antigen determined by the MTT assay, and the H³-thymidine uptake test. The SI obtained was similar; 1.7 for the MTT and 1.5 for the H³-thymidine uptake. Results suggest that the MTT bioassay and frozen cells can be used as alternatives in the in vitro bovine lymphocyte proliferation test.

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