Gadolinium chloride gdcl3

The membrane “rigidifier” Dimethylsulphoxide (DMSO, 2% v/v; Roth, Karlsruhe, Germany), and the membrane “fluidiser” Benzylalcohol (BA, 10 mM, Roth, Karlsruhe, Germany) were used to modulate plasma-membrane fluidity^{66 (link)–68 (link)}. The microtubule compounds taxol and oryzalin were employed to stabilise or disrupt microtubules, respectively^{38 (link)}. Diphenyleneiodonium chloride (DPI, 100 µM; Sigma-Aldrich, Deisenhofen, Germany) was used to inhibit apoplastic oxidative burst by the plasma membrane located NADPH oxidases^{69 (link)}, and Gadolinium chloride (GdCl₃, 100 µM; Sigma-Aldrich, Deisenhofen, Germany) to block the calcium channels^{66 (link)}. All inhibitors were diluted from a stock solution in DMSO, the maximal concentration of the solvent was 0.1%, and the experimental design, therefore, included one set with 0.1% DMSO as solvent control. To probe for the effect of microtubule stability, cells were treated with 10 μM taxol (Sigma-Aldrich, Deisenhofen, Germany) prior to elicitation by harpin or flg22. To eliminate microtubules, we added 10 μM of oryzalin (Sigma-Aldrich, Deisenhofen; Germany) 1 h prior to elicitation. A solvent control with 0.1% DMSO was included throughout.

Adult male C57BL/6 mice were obtained from Sipper BK Experimental Animals, Co. (Shanghai, China). IL-10 knockout mice (C57BL/6 background) were purchased from Jackson Laboratory (Bar Harbor, ME, United States). All mice were housed under SPF facility in a 12:12 light–dark cycle and used for animal experiments at age of 6–8 weeks. All procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of the Second Military Medical University. Mouse recombinant IL-35 was purchased from AdipoGen (Liestal, Switzerland). LPS (Escherichia coli, O26:B6), D-GalN, gadolinium chloride (GdCl₃), 7-AAD, and bovine serum albumin (BSA) were from Sigma (St. Louis, MO, United States). Fluorescein-conjugated mAbs against Annxin V and F4/80 were obtained from BD-PharMingen (San Diego, CA, United States). Sources of the other antibodies and kits are described in the methods below.

Endothelial cell medium was purchased from AllCells (Emeryville, CA, USA). Bovine serum albumin (BSA), poly-d-lysine, ethylenediaminetetraacetic acid (EDTA), lipopolysaccharide (LPS), Triton X-100, 4′,6-diamidino-2-phenylindole (DAPI), 3,3-diamino-benzidine tetrahydrochloride (DAB), gadolinium chloride (GdCl₃) and annexin V were obtained from Sigma-Aldrich (St Louis, MO, USA). 0.25% Trypsin-EDTA was from Gibco (Grand Island, NY, USA). CellTracker Green CMFDA and CellTracker Red CMTPX were from Invitrogen (Carlsbad, CA, USA). Lactadherin was prepared in our laboratory. Human factors Va, VIIa, VIII, IXa, X, Xa, prothrombin and thrombin were all from Haematologic Technologies (Burlington, VT, USA). The Chromogenix substrates S-2238 and S-2765 were from DiaPharma Group (West Chester, OH, USA). Monoclonal antibodies against CD42b (glycoprotein Ib, clone HIP1), CD62P (P-selectin, clone AK-4), CD41a (clone HIP8), CD45 (clone H130), CD36 (glycoprotein IV, clone CB38) were from Becton Dickinson Biosciences (San Jose, CA, USA). Monoclonal antibody against αvβ3 integrin was from Abcam (ab7166), Labome (clone LM609), and polyclonal antibody from Bioss (bs1310R). Alexa Fluro 488 or 647-conjugated CD41a and Alexa Fluro 647-labeled CD45 were prepared in our laboratory. FITC-labeled monoclonal antibody against CD62P was from Abcam (ab6632, Cambridge, MA, USA).

S. aureus (MFP03) and S. epidermidis (MFP04) were collected from the skin of healthy volunteers46 (link). These bacteria were characterized by phenotypic, metabolic, MALDI-Biotyper proteomic analysis and 16S ribosomal RNA gene sequencing. All bacteria were grown at 37 °C in Luria-Bertani (LB) medium under gentle agitation (180 rpm). Bacteria were stored on cryobeads at −140 °C and subjected to two pre-culture phases. For the studies, bacteria collected at the end of the exponential growth phase were diluted in fresh broth. The peptides, diluted in sterile physiological water (NaCl 0.9%), or an equivalent volume of physiological water in control studies, were added at the beginning of the log growth phase. Before the tests, the bacteria were harvested by centrifugation (7,000 × g) and washed with sterile physiological water to remove any trace of free peptide. The bacterial density and the absence of contamination were controlled by plating. The viability of the bacteria in eukaryotic cell culture medium and under different culture conditions was controlled in preliminary studies (data not shown). CGRP and Substance P were obtained from Polypeptides (Strasbourg, France). Gadolinium chloride (GdCl₃) was obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France).