Figure 1: Confocal immunofluorescence assessment of nuclear translocation of (p65) NFκB in MSCs following stimulation with LPS. ((a)–(c)) Overlay of projections of nuclei (blue channel) and (p65) NFκB (red channel) in MSCs. (a) Control cells. (b) MSCs challenged with 500 ng/mL LPS for 1 h. Activation of (p65) NFκB nuclear translocation was defined by an increase in immunofluorescence of (p65) NFκB in the nuclear regions. Nuclear regions of MSCs were determined by counterstaining of nuclear DNA with Hoechst 33342 (blue channel). Nuclear localization of (p65) NFκB appears in purple (indicated with arrows). (c) is the same as (b) except that MSCs were preincubated with 10 μM PDTC (pyrrolidine dithiocarbamate), an inhibitor of NFκB. (d) The treatments were the same as in (c) but the presented image is overlay of projections of Annexin V (green channel), a marker of proapoptotic transformations, and a respective DIC image of MSCs. Apoptotic events are indicated with arrows. The confocal images were taken with pinhole setup to obtain 0.5 μm Z-sections. Conditions: MSCs were incubated with 500 ng/mL LPS in the medium either 1 h or 3 h (see Section 2). (e) Histogram depicting increase in frequency (per 100 cells) of occurrence (p65) NFκB nuclear translocation in MSCs treated with LPS. Spatial appearance of (p65) NFκB in MSCs was determined with immunofluorescence confocal microscopy as indicated in Section 2. Confocal projections of nuclear fractions of (p65) NFκB are shown in ((a)–(c)).