HeLa cells were either heat-shocked or stimulated with anisomycin (a protein synthesis inhibitor and activator of stress-activated protein kinases) to induce a stress response. Cells were grown to ~80% confluency at 37ºC, heat shocked at 43ºC for two hours, and returned to 37ºC for 18 hours. Alternatively, cells grown to confluency were treated with 1 µg/mL anisomycin for 15 minutes. Both heat-shocked and anisomycin-treated cells were harvested by scraping, lysed with Cell Lysis Buffer 3, and adjusted to a protein concentration of 1 mg/mL. The Accuri C6 Flow Cytometer System (Accuri Cytometers) was utilized to analyze tissue culture lysates with the Heat Shock Protein/Chaperone 8-Plex MultiBead Kit in 96-well filter plates, according to kit instructions.
Recombinant standards of known concentrations were used to generate standard curves utilizing MultiBead software. The average mean fluorescent intensities (MFIs) from duplicate wells are shown in Figure 2A (ng/mL) and in Figure 2B (units/mL) against which MFIs of cell lysates were plotted to calculate HSP protein concentration.