Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios

Fish Shellfish Immunol. 2003 Sep;15(3):225-40. doi: 10.1016/s1050-4648(02)00160-2.

Abstract

A flow cytometric method to measure the production of oxidative metabolism products was adapted for use with Crassostrea gigas hemocytes. The method is based upon the oxidation, by hydrogen peroxide (H2O2), of intracellular 2',7'-dichlorofluorescin (DCFH) to green-fluorescent dichlorofluorescein. Activation of the respiratory burst (RB) was tested using phorbol myristate acetate with no success. By contrast, activation by zymosan particles increased oxidation of DCFH in C. gigas hemocytes, mainly granulocytes, and optimization tests showed a good response with 20 zymosan particles per hemocyte. Anti-aggregant solution, used to prevent hemocytes from clumping during bleeding, inhibited the RB activity measured by DCFH oxidation. The flow cytometric method developed during this work was used to evaluate the DCFH oxidation-inhibiting capacity of four strains of vibrio bacteria, known or suspected to be pathogenic for bivalves.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Flow Cytometry
  • Fluoresceins / metabolism
  • Hemocytes / metabolism*
  • Hydrogen Peroxide / metabolism
  • Ostreidae / cytology*
  • Ostreidae / metabolism
  • Vibrio / pathogenicity*

Substances

  • Fluoresceins
  • 2',7'-dichlorofluorescein
  • Hydrogen Peroxide