Figure 3.

Neutralized medium prevents apoptosis. Conditioned medium was generated as described in Figure 2. (a, top) DNA ladders from the cardiac myocytes used to generate the spent medium. (a, bottom) Spent medium was added directly to fresh plates of cardiac myocytes (middle 2 lanes), or it was neutralized to pH 7.4 with HEPES (20 mM final concentration) and NaOH and then added to a second set of fresh cardiac myocytes. Both sets of plates were incubated under hypoxia for 24 hours and analyzed for DNA fragmentation. Control plates were incubated under aerobic or hypoxic conditions in parallel. (b) Parallel sets of cardiac myocytes exposed to hypoxia without medium change. In the first set (b, left), the acid was allowed to accumulate exactly as described in Figure 1b; in the second set (b, right) aliquots of 250 mM HEPES and 250 mM NaOH were added every 12 hours to maintain a [pH]_o of approximately 7.1. Measurements of DNA fragmentation and determinations of percent condensed nuclei (c) were as described in Methods. In all cases, the medium pH was measured immediately before the cultures were harvested. (d) Typical field of myocytes stained with HOECHST 33342 and anti-myosin antibody as described in Figure 1d. Results in a, b, and c are from typical experiments; error bars in c are SEM (n = 3).