Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Rac1 regulates platelet shedding of CD40L in abdominal sepsis.

doi: 10.1038/labinvest.2014.92

Figure Lengend Snippet: Figure 3 CXCL2 induces Rac1 activity in neutrophils. (a) Neutrophils were isolated by magnetic beads and then 1 106 neutrophils were incubated with CXCL2 (0.3 mg/ml) for 20 min with and without Rac1 inhibitor NSC23766 (N6-[2-[[4-(diethylamino)-1-methylbutyl] amino]-6- methyl-4-pyrimidinyl]-2 methyl-4, 6-quinolinediamine trihydrochloride; 10 mM). Active Rac1 protein was pulled down from neutrophil lysates by using GST-PAK beads. Rac1-GTP was detected by western blot. (b) Band intensities were quantified by densitometry and normalized to total Rac1. Bars represent mean±s.e.m. and n ¼ 4. *Po0.05 vs control and #Po0.05 vs vehicle þ CXCL2.

Article Snippet: About 1 106 neutrophils were preincubated with NSC23766 (10 mM) for 20 min before challenging with 0.3 mg/ml recombinant mouse CXCL2 (R&D Systems) or PBS as a control for 30 min at 37 1C.

Techniques: Activity Assay, Isolation, Magnetic Beads, Incubation, Western Blot, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Rac1 regulates platelet shedding of CD40L in abdominal sepsis.

doi: 10.1038/labinvest.2014.92

Figure Lengend Snippet: Figure 5 Rac1 regulates neutrophil secretion of matrix metalloproteinase-9 (MMP-9). (a) Neutrophils were isolated by magnetic beads. (a) Isolated neutrophils were incubated with CXCL2 (0.3 mg/ml) and then was the level of MMP-9 in permeabilized neutrophils determined by confocal microscopy. (b) Summarized data showing mean fluorescence intensity (MFI) of MMP-9 in neutrophils. Bars represent mean±s.e.m. and n ¼ 4. *Po0.05 vs control and #Po0.05 vs vehicle þ CXCL2.

Article Snippet: About 1 106 neutrophils were preincubated with NSC23766 (10 mM) for 20 min before challenging with 0.3 mg/ml recombinant mouse CXCL2 (R&D Systems) or PBS as a control for 30 min at 37 1C.

Techniques: Isolation, Magnetic Beads, Incubation, Confocal Microscopy, Fluorescence, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Rac1 regulates platelet shedding of CD40L in abdominal sepsis.

doi: 10.1038/labinvest.2014.92

Figure Lengend Snippet: Figure 6 Neutrophil-derived matrix metalloproteinase-9 (MMP-9) regulates platelet shedding of CD40L. Isolated MMP-9 gene-deficient platelets were incubated with proteinase-activated receptor-4 (PAR4) and with supernatants from either wild-type or MMP-9-deficient neutrophils stimulated with CXCL2 for 30 min. Soluble CD40L was determined in the supernatants by enzyme-linked immunosorbent assay. Nonstimulated platelets served as control. Bars represent mean±s.e.m. and n ¼ 4-5. *Po0.05 vs control, #Po0.05 vs control, and ¤Po0.05 vs wild-type neutrophil supernatant.

Article Snippet: About 1 106 neutrophils were preincubated with NSC23766 (10 mM) for 20 min before challenging with 0.3 mg/ml recombinant mouse CXCL2 (R&D Systems) or PBS as a control for 30 min at 37 1C.

Techniques: Derivative Assay, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Control