Demonstration of Platelet Electrophoretic Mobility Slowing Factor (Psf) in Supernatants of Human Lymphocyte Cultures Stimulated by Phytohaemagglutinin (Pha)

Abstract

374 MATERIAL AND METHODSLymphocytes: Blood lymphocytes were obtained from normal donors by centrifugation in a Ficoll-Hypaque gradient (i), and adjusted to a cell concentration of 19 X loVml. This preparation contained 90 per cent lymphocytes and 10 per cent monocytes.Preparation »/ lupernatants from PH.1-activated and rontro! cultures of lymphocytes (2, 3) : A stock solution of PHA was prepared by adding 5 ml sterile distilled water to freeze-dried PH.^ (phvtohaemagglutinin, HA 15.5 ml dried, Wellcome Reagents Ltd., Beckenham, England). Two milliliters TC medium 199 were mixed with 500 /il of the PHA stock solution. From this mixture, 15 ix\, containing 30 Mg of PHA. were added to 135 ^1 of the suspension of lymphocytes. As control, 135 ftl of the suspension of lymphocytes were added to 15 M1 of a mixture composed of 500 /il sterile distilled water and 2 ml TC 199.The cell suspensions were incubated for 1 h at 37° C. Then the cells were sedimented by centrifugation at 220 g for 5 min, washed three times in Hank's balanced salt solution (HBSS) and resuspended in i ml TC 199 without serum. Both the cell suspensions which had a cell concentration of 2 X lo^/ml, were divided into three aliquots of 300 fil and cultured for 2 days in an atmosphere of 5 per cent COs and 95 per cent air.Cell-free supernatants were collected by centrifugation at 3000 r.p.m. for ro min at the end of the culture. PHA was added to control supernatants in original amounts, after harvesting.Chrnmatographic fractinnation: To provide sufficient material for chromatography, 50 ml each of control and active supernatants were concentrated lo-fold hy pressure dialysis.Each supernatant (s ml) was applied to a column (1,6 X 70 cm), packed with I'ltrogel .^C a 44, equilibrated with sterile, endotoxin-free isotonic saline and precalibrated with BSA (8 mg; moi. wt. 69,000), egg albumin (8 mg; moi. wt. 45,000) and lysozyme (8 mg; moi. wt. 14,000). The column was eluted at a flow rate of 12 ml/h. The eluates were pooled in four fractions, which were concentrated 20-fold by pressure dialysis before use.Fraction I contained material eluted before BSA, fraction II eluted as BSA, fraction III eluted as egg albumin and fraction IV eluted as lysozyme.Each chromatography was performed in triplicate. Platelet electrnphoretic mobility (PEM) was determined by means of a Zeiss Cytopherometer (12).Platelet rich plasma (PRP): Human blood was obtained from healthy donors by tapping nine parts of blood from an antecubital vein into one part of sodium citrate {3.8 per cent according to Westergreen).Platelets wert counted in a Burker chamber by the Palumbo-Dini method (16) and adjusted to a density of 3 X io'/25o ^g of active and control chromatographic fractions. These mixtures were immediately placed, without incubation.