Details

Autor(en) / Beteiligte

• Oliver, Cynthia N.

Titel

[60] Measurement of oxidized proteins in systems involving activated neutrophils or HL-60 cells

Ist Teil von

• Methods in Enzymology, 1990, Vol.186, p.575-579

Ort / Verlag

San Diego, CA: Elsevier Science & Technology

Erscheinungsjahr

1990

Quelle

MEDLINE

Beschreibungen/Notizen

• This chapter describes the measurement of oxidized proteins in systems involving activated neutrophils or HL-60 cells. The measurement of oxidized proteins can carried out with either freshly isolated neutrophils or HL-60 cells, but HL-60 cells are used at densities 2–3 times greater than that of neutrophils because of lower respiratory burst activity. Assays must be adapted for each in vitro system depending on the cells or proteins used and the information required. For example, when oxidized proteins and enzymatic activity are determined in activated neutrophils alone, 108 to 109 cells/ml are suspended in an isotonic buffer with 1 mM CaCl2, 1 mM MgC12, and 1 mg/ml glucose and stimulated with 100 ng of phorbol 12-myristate 13-acetate (PMA). For these experiments, cells are isolated from leukophoresis preparations enriched for neutrophils, and although high yields of cells are obtained by this method, the levels of spontaneous activation are higher than cells obtained from whole blood without prior leukophoresis. Following an incubation of 30 minutes to 1 hour, neutrophils are collected by centrifugation and disrupted by sonication using a Heat Systems Sonifier Cell Model 185 equipped with a microtip. Insoluble cellular debris is removed by centrifugation, and oxidized protein is determined by 2,4-dinitrophenylhydrazine reactivity using the filter paper method. Purified proteins and enzymes are incubated with the activated neutrophils of HL-60 cells to determine the possible susceptibility for neutrophil-mediated oxidative modification in vivo.