COMPARISON OF FLOW-CYTOMETRY AND WESTERN BLOTTING TO MEASURE HSP70

The levels of constitutive and inducible forms of heat shock protein 7 0 (hsp73 and hsp72, respectively) through the cell cycle were measured in CHO cells by flow cytometry and Western blotting at various times after heating, Cells were labelled with antibody C92 (hsp72) or N27 (h sp73) and propidium iodide prior to analysis by flow cytometry. Cells were heated for 15 min at 45 degrees C, then analysed from 3 to 36 h l ater, There was about a tenfold increase in hsp72 in early S phase cel ls beginning within 6 h after heating and these cells gradually cycled though S phase so by 36 h most of them had divided, When CHO cells we re exposed to 10 mu M sodium vanadate, an inhibitor of tyrosine phosph atase, for 24 h prior to heating, the induction of hsp72 in early S ph ase cells was almost completely inhibited. Heated cells did not expres s hsp73 in a cell-cycle-dependent manner, Hsp73 increased uniformly in all cells by 10 h after heating and sodium vanadate did not affect th e expression. Quantitative comparisons of the relative levels of hsp72 and hsp73 measured by how cytometry and Western blotting were in exce llent agreement. Control and heated cells were labelled with Hoechst 3 3342 and sorted from G1, S, and G2/M phases and processed by Western b lotting to verify the cell cycle dependent increase in hsp72 as measur ed by now cytometry, Again there was excellent agreement between the W estern blotting and flow cytometry results. (C) 1996 Wiley-Liss, Inc.