Acta Biologica 40. (1989)
1-2. szám - F. Liszt–K. Jobst: Luminol dependent chemiluminescence of quartz and zymosan stimulated neutrophils
122 F. LISZT and K. JOBST some cases the protein pattern of zymosan stimulated PMNL was investigated by high resolution two dimensional electrophoresis. This way we could decide how this stimulus might cause specific changes in the pattern of synthetized protein during phagocytic process to reveal the short term effects. Materials and Methods Heparin anticoagulated (10 U/ml) venous blood from healthy donors was obtained. The cells were isolated by a modified procedure according to Hjort and Johnson /5/ on a two step discontinuous (55—75%) Percoll gradient. After centrifugation the distribution of the cells was 75—85% PMNL and 25—15% lymphocytes, the viability was 95—98%. The PMNL were resuspended in Hanks solution and the count was adjusted to 6xl0°/ml. We used DQ—12 quartz (FRG locality.of Dörentrup) with diameter of 2—5 yum. Zymosan was purchased from Serva (Heidelberg, FRG) with diameter of 4—7^um. Calcium ionophore (Boehringer Mannheim, FRG) was applied in a final concentration of 1 /jmol/1. Serum treated quartz and zymosan were prepared by incubating quartz and zymosan particles at a concentration of 50 mg/ml with 50% fresh pooled serum in Hanks solution at 37 UC for 30 min . The mixtures were centrifuged for 10 min and then washed twice with Hanks solution before being resuspended to the same concentration in Hanks solution. For the luminol dependent chemiluminescence study 100 ul of the PMNL suspension, 500 yjl of 0.2 mmol/1 luminol and 1.5 ml of Hanks solution were preincubated for 10 min at 37 °C. The reaction was initiated by adding 20 ul particle suspension. A control without particle stimulation was carried out to exclude the inducing effect of luminol. CL was measured by an XP 2020-Q photomultiplier tube (Applied Photophysics, England) coupled to an ICA 70 (KFKI, Budapest, Hungary) multichannel analyzer and Biolumat 9505 analyzer (Laboratorium Prof. Dr. Berthold, Wildbad, FRG — generous gift of the Alexander von Humboldt Foundation). In parallel with the CL measurements, the protein pattern of zymosan stimulated PMNL was investigated by high resolution two dimensional polyacrylamide gel electrophoresis. Isolated PMNL were incubated in methionine deficient MEM Dulbeeco medium supplemented with fetal bovine serum (5 ml/l) and 40 juCi of y S)-methionine. Approximately 5x10^ cells with 20 jjI zymosan suspension were cultured per well in flat bottom multiwell tissue culture plates. Cultures with and without zymosan were incubated for 18 h at 37 C in humidified atmosphere containing 5% C0„. At the end of the labeling period cells were harvested by brief centrifugation in a Beckman Microfuge and the pellets were lysed in O'Farrel lysis buffer /7/. After centrifugation at 100 000 g for 30 min the lysed samples were analyzed by high resolution two-dimensional polyacrylamide gel electrophoresis /7/. The protein spots were visualized by fluorography using KODAK RP Royal X-0mat film /2/.