Comparison of Cul Tures of Human Lymphocytes Obtained Following Nh4Cl Induced Red Blood Cell Lysis and Ficoll-Hypaque Density Gradient Centrifugation

Autor: Simon, M. R., Roi, L. D., Desai, S., Salberg, D. J., Rosec, N. R.
Zdroj: Immunological Investigations; 1983, Vol. 12 Issue: 3 p301-314, 14p
Abstrakt: Lymphocyte thymidine uptake in leucocytes prepared by NH4Cl induced RBC lysis was compared with that of lymphocytes obtained following Ficoll-Hypaque centrifugation using polynomial regression dose response curves which adjusted for inherent subject variability. Eleven male subjects were studied. Heparinized blood was either layered on- Ficoll-Hypaque or treated with 0.87% NH4Cl to induce RBC lysis. Phytohemagglutinin-P (PHA) dose response curves were determined for each subject using triplicate 5 day cultures. For each subject, at each PHA concentration ranging from 12.5 to 167ug/ml, triplicate mean cpm of Ficoll-Hypaque treated lymphocytes and NH4C1 treated lymphocytes were compared by the 2 tailed Student's T test. Responses of cultures of NH4Cl treated lymphocytes were significantly different (<0.05) from the responses of cultures of Ficoll-Hypaque treated lymphocytes for at least 2 PHA doses in 8 of the 11 subjects. The quadratic dose response curves were also significantly different (p<0.05) for 9 of the 11 subjects. A repeated measures quadratic regression was used to determine the best fit dose response curves for each of the experimental conditions. For Ficoll-Hypaque treated-lymphocytes, mean cpm=44,977+1400.4 (PHA concentration)-5.90 (PHA concentration) 2+ subject effect; s.e.=22,256; for NH.C1 treated lymphocytes, mean cpm=58,039+1022.8(PHA concentration)-5.32 (PHA concentration)2+ subject effect; s.e.=37,966. The p values from the F tests for significance of these quadratic regressions are <0.0001 and >0.28, respectively. The s. e. of the quadratic regressions are different, F=2.91, d. f.=30,30; p<0.001. The lack of significance of the NH4Cl treated lymphocytes1 dose response quadratic regression and its larger s. e. indicates that the NH4C1 treated lymphocyte culture dose response curve is less predictable than that of Ficoll-Hypaque treated lymphocytes.
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