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Figure 1 | Critical Care

Figure 1

From: Increased HMGB1 expression and release by mononuclear cells following surgical/anesthesia trauma

Figure 1

Analysis of HMGB1 cellular expression. (a) Flow cytometric analysis of HMGB1 expression in monocytes from one patient and one control subject (healthy donor). Mononuclear cells were drawn from the patients at three different times, that is, t0: before surgery, t1: immediately after surgical procedure; t2: at 24 hours following intervention. Cells were stained with polyclonal anti-human HMGB1 1 μg/ml (Abcam) for one hour at room temperature. Nonspecific binding was determined by an unlabeled isotypic control antibody (Coulter-Immunotech, Hamburg, Germany). After washing with cold PBS, cells were incubated with FITC-conjugated anti-rabbit IgG and then analyzed by flow cytometry. Antibody reactivity was reported as mean fluorescence intensity. Histograms show the log fluorescence versus the cell number. (b) Results of flow cytometric analysis of HMGB1 expression in monocytes from controls (healthy donors) and from the patients under test at three different times: t0 = before surgery, t1 = immediately after surgical procedure; t2 = at 24 hours following intervention. Mean fluorescence intensities were measured and plotted values represent mean ± SD. ***t1 vs t0, t1 vs t2: P < 0.0001. (c) Monocytes cells were sampled at the indicated time points and subjected to nuclear (N) and cytoplasmic (C) fractionation. The levels of endogenous HMGB1 in the nuclear and cytoplasmic fractions were determined by immunoblotting with anti-HMGB1 antibodies. Laminin B served as nuclear contamination marker and α-tubulin as cytoplasmic contamination marker. Protein loading within each compartment was also normalized with Laminin B and α-tubulin, respectively.

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