The kinetics of HMGB1 release by necrotic cells. Jurkat cells in serum-free medium were induced to undergo by freeze-thaw, heat, H2O2, ethanol or no treatment (controls), and incubated at 37° for the times indicated. Culture media were concentrated and then examined by Western blotting with protein detection by ECL. As controls for HMGB1 detection,100 ng, 50 ng and 25 ng of recombinant hHMGB1-histidine tagged protein were analyzed. Figure is a digital image of a blot taken with an Alpha Innotech digital imager.