Important Variables in Granulocyte Chemiluminescence

Abstract

Summary

A CL system consisting of canine granulocytes stimulated with opsonized zymosan was used to examine factors which lead to variability in light production. Variability of CL response was associated with granulocyte aggregation. Granulocyte aggregation was reduced by: (i) reducing centrifu-gation and mixing forces used to prepare cell suspensions, (ii) preparing cell suspensions at 4° in Ca²⁺- and Mg²⁺-free cell media containing divalant cation chelating agents, and (iii) lysing red blood cells by the ammonium chloride technique.

The type of cell media used during the CL run was another important variable. Solutions containing complex mixtures of amino acids and vitamins gave markedly reduced levels of CL. Tryptophan, ascorbic acid, iron salts, and all proteins tested caused significant suppression of CL. Glucose, Ca²⁺, and Mg²⁺ in the final reaction mixture were necessary for maximal CL response. The optimal cell media for CL, therefore, was buffered salt solution containing glucose, Ca²⁺, and Mg²⁺, but not amino acids, vitamins, proteins, or iron salts.

The authors gratefully acknowledge the helpful suggestions of Drs. Liana Harvath and Robert Epstein. This work was supported by West Side VA Medical Center MRIS 0394-08 funds.