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 markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased 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 response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.
Laboratoire de Physiologie Respiratoire, UFR Cochin Port-Royal, Paris, France.
M
M
Bachelet
E
E
Mariéthoz
N
N
Banzet
E
E
Souil
F
F
Pinot
C Z
CZ
Polla
P
P
Durand
I
I
Bouchaert
B S
BS
Polla
D015153:Blotting, Western; D002478:Cells, Cultured; D005434:Flow Cytometry; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D007124:Immunoenzyme Techniques; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013312:Stress, Physiological / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins