Fig. 4.

Detection of Hsp60 on the surface of differentiating megakaryocytes and senescent platelets. a MEG-01 cells were pooled from several days under standard culture conditions and analysed for Hsp60 expression with FITC-conjugated secondary antibodies and flow cytometry. Viability (V) of the cells was verified through exclusion of PI. b Flow cytometry confirms surface expression of Hsp60 in differentiating MEG-01 cells, but minimal surface expression on viable cells. Differentiating MEG-01 cells which stain weakly for PI exhibit strong expression of Hsp60 on the surface. c In late stages of differentiation and apoptosis, MEG-1 cells have scant cytoplasm. Coincident with strong PI staining of their nuclei, surface expression of Hsp60 is reduced. d, e Senescent platelets produced by standard cultures of MEG-01 stain for FITC-conjugated annexin V, indicating PtdSer exposure on the platelet surfaces. f, g Senescent platelets (which stain for annexin V) express Hsp60 on the surface. Hsp60 surface expression and PtdSer externalization are thus common events in platelet ageing and apoptosis. h Terminal differentiation of human megakaryocytic MEG-01 cells culminates in release of platelets through protrusions in the cell membrane, which resemble apoptotic membrane blebbing (bright-field image). Hsp60 (FITC-conjugated antibody secondary detection) and calreticulin (TRITC) appear to be segregated to different intracellular locations. Hsp60 is distributed to the outer cortex of the cytoplasm and membrane blebs (arrow), whilst calreticulin is limited to the inner cytoplasm. (bar 10 μm)