Use of different human myeloid leukemia cell lines to determine the specificity of 3,3’-diaminobenzidine cytochemistry for the ultrastructural localization of myeloperoxidase and catalase

Abstract

Myeloid cells are known to contain myeloperoxidase (MPO) and catalase. This study has used MPO and catalase replete and deficient myeloid cell lines to clarify the localization of these components using 3,3’-diaminobenzidine (DAB) ultrastructural cytochemistry. Conditions of DAB incubation can be modified to preferentially stain catalase (alkaline at pH 9.7) or MPO (neutral at pH 7.0-7.6), but crossreactivity persists, preventing the discrimination between catalase and peroxidase. Biochemical assays demonstrated both MPO and catalase in HL60 cells; similar amounts of catalase but no MPO activity in the A7 cell line; increased amounts of catalase but no MPO activity in the HP50 and HP100 cell lines; and neither MPO nor catalase in the KG1 cell line. Neutral DAB stained MPO (pH 7.4; [DAB] 5 or 20 mg/10 mL 0.05 M Tris; 30 min or 120 min; 24° or 37°C; 0.01% H2O2) in HL60 (Fig. 1), but not in A7. Alkaline DAB intensely stained catalase (pH 9.7; 20 mg/10 mL; 120 min; 37°C; 0.01% or 0.03%) in A7.