FLOW-CYTOMETRY IS A RAPID AND RELIABLE METHOD FOR EVALUATING HEAT-SHOCK-PROTEIN-70 EXPRESSION IN HUMAN MONOCYTES

The increasing interest in stress/heat shock proteins (Hsps) as marker s of exposure to environmental stress or disease requires an easily ap plicable method for Hsp determination in peripheral blood cells. Of th ese cells, monocytes preferentially express Hsps upon stress. An appro priate fixation/permeabilization procedure was developed, combined wit h immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Highe r relative fluorescence intensity was observed in cells exposed to hea t shock (HS), reflecting a higher expression of Hsp70 in these cells a s compared with cells kept at 37 degrees C. The heat-inducible increas ed Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in resp onse to HS. Simultaneous measurements of intracellular Hsp70 and membr ane CD14 expression revealed that the higher Hsp70 inducibility coinci ded with the higher CD14 expression. Comparisons performed with biomet abolic labelling, Western blotting, immunofluorescence and immunoperox idase microscopic analysis, showed a high concordance between these di fferent methods; however, cytometry was more sensitive for Hsp70 detec tion than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited f or the determination of protein levels in individual cells from an het erogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.