Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biometabolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformaldehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve, followed by permeabilization with Triton X-100 (0.1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1.2-fold increase in Hsp70/Hsc70 accumulation (P < 0.001) in protoplasts, while Western blotting, quantified by image analysis, showed induction under similar conditions but at lower significance (P < 0.05). The coefficients of variance for flow cytometry and Western blotting were 30.7 and 49.8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 degrees C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.
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