Quantitative analysis of apoptosis in PBMCs.

<p>Representative distributions of the fluorescence intensity of annexin-V-FITC and PI binding of (a) EN (n = 10) and (b) CP (n = 5) monocytes after 24-h stimulation with rWmhsp60 and CHX. Early apoptotic cells (E) were annexin-V-FITC⁺/PI⁻, late apoptotic cells (L) were annexin-V-FITC⁺/PI⁺ and dead cells (D) were annexin-V-FITC⁻/PI⁺. The scatter plot represents the percentage of early, late apoptotic and dead cells in (c) EN and (d) CP monocyte populations. Values are represented as mean ± S.D and “*” denotes p<0.05. rWmhsp60 induces apoptosis in monocytes of EN via caspase cascade activation. (e) rWmhsp60- and CHX-treated monocytes from EN were harvested, and the levels of caspase-3, caspase-9, p53 and PARP were determined by Western blot analysis as described in Methods section with the respective cleaved and total antibodies. (f) Bar graph represents the fold change of normalized IOD of the respective blots. (g) Monocytes of EN were cultured in the presence of CHX and rWmhsp60 for 24 h and determined the caspase-3 activity using the fluorogenic substrate Ac-DEVD-AMC as detailed in Methods section. The activity expressed as % fold increase in relative fluorescence units (RFU) (mean ±S.D, n = 10) and “*” represents p<0.05.