(A) Intraperitoneal cell fractions characterised by flow cytometry in controls (green) and 10 hr after caecal ligation and puncture (CLP, orange) in a tSNE plot of viable (AmCyanlow) CD45high cells. …
(A) Flow cytometry gating strategy for inflammatory cells isolated from the peritoneal cavity. Identified peritoneal macrophages subsets in controls versus 10 hr after caecal ligation and puncture …
(A) Schematic representation of our conditional Connexin-43 KO mouse model (MAC-CX43 KO). (B) Gja1 (Cx43) mRNA expression in LPS (1 µg/ml)-stimulated peritoneal macrophages isolated from MAC-CX43 KO …
(A) Flow cytometry gating strategy for human inflammatory cells isolated from fluids obtained from peritoneal lavage during abdominal operations. Macrophages were defined as viable (AmCyanlow), CD45h…
(A) Increased expression of CX43 in peritoneal macrophages after 5 hr LPS stimulation (1 µg/ml). (B) Single channel staining for PE-Texas Red-labelled CX43, FITC-labelled F4/80 and DAPI.
(A) Elevated CX43 protein expression in the liver 5 and 10 hr after CLP compared to controls (N = 3 animals in each control group from the gels at 5 hr and 10 hr, all used for the calculation of the …
(A) Expression of CX43 in the liver 10 hr after CLP. Data are representative of >3 experiments. (B–C) Following CLP (orange), CD8 positive cytotoxic T cells decreased in the liver, while NK cells …
(A) Extracellular ATP levels as assessed by luminescence of i.p. injected 5 × 106 HEK293-pmeLUC D-luciferin activated cells (in counts per second (cps)). Luminescence was significantly higher 10 hr …
(A–B) Quantification of functional markers of M1 macrophage differentiation Inos and Il12rb, in peritoneal macrophages with CX43 blocking or deletion by qPCR (unpaired t-test)*. (C–E) Markers of M2 …
(A–B) TNF-alpha release from peritoneal macrophages in response to LPS (1 µg/ml) and by blocking specifically purinergic receptors including P2X (A) and P2Y (B) receptors (N = 4)*. (C) Purinergic …
(A–B) CD73 and CD39 expression was assessed by qPCR (each dot is representative of a single independent biological samples, unpaired t-test). (C–D) Kinetic of extracellular ATP degradation from WT …
(A–B) Clinical outcome using Murine Sepsis Score and weight loss following CLP (N = 5 animals per group, two-way ANOVA). (C) Survival of mice following caecal ligation and puncture (CLP). Mice …
(A–B) No difference in bacterial load as determined by CFUs after serial dilution of blood and peritoneal fluid 10 hr post CLP (N = 4 animals per group, Mann-Whitney-test).
(A) Schematic representation of clustering after CyTOF analysis and dot plot representation of tSNE analysis showing different clusters in the peritoneal cavity 10 hr after CLP compared to control …
(A–B) Impact of CX43 on T-lymphocyte recruitment in the peritoneal cavity, CD4 positive T helper and CD8 cytotoxic T cells were similar between MAC-CX43 KO and WT mice (N = 5 animals per group, …
(1) Following caecal ligation and puncture (CLP), bacteria invade the peritoneal cavity. (2) Bacteria-derived products, for example LPS, interact with TLR-4 to increase CX43 synthesis in a …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Anti-Connexin-43 (rabbit, polyclonal) | Cell Signalling | 3512, RRID:AB_2294590 | WB (1:850) IHC/IF (1:100) |
Antibody | Anti-β-actin HRP-conjugated (mouse, monoclonal) | Sigma-Aldrich | A2228, RRID:AB_476697 | (1:5000) |
Antibody | Anti-rabbit HRP-conjugated (goat, polyclonal) | Dako | P0448, RRID:AB_2617138 | (1:5000) |
Antibody | Anti-rabbit biotinylated (goat, polyclonal) | Dako | E0432, RRID:AB_2313609 | (1:200) |
Antibody | Anti-rabbit IgG FITC-conjugated (goat, polyclonal) | Sigma-Aldrich | F0382, RRID:AB_259384 | (1:200) |
Cell line (Homo sapiens) | HEK293-pmeLUC | doi:10.1371/journal.pone.0002599 | ||
Chemical compound | ARL 67156 | Sigma-Aldrich | 67156 | |
Chemical compound | TRIzol | Roche | 11667165001 | |
Chemical compound | Omniscript RT Kit 200 | Quiagen | 205113 | |
Chemical compound | Lipopolysaccharides E.coli O111:B4 | Sigma-Aldrich | L4130 | |
Chemical compound | Pam3CSK4 | Invivogen | tlrlpms | |
Chemical compound | 18-alpha- glycyrrhetinic acid | Sigma-Aldrich | G8503 | |
Chemical compound | Gap27 | Tocris-Bioscience | 1476 | |
Chemical compound | Probenecid | Sigma-Aldrich | P8761 | |
Chemical compound | Carbenoxolone | Sigma-Aldrich | C4790 | |
Commercial assay | ATP Reagent SL | BioThema | 144–041 | |
Commercial assay | Resazurin assay | Sigma-Aldrich | 199303 | |
Commercial kit | V-PLEX Mouse Cytokine 19-Plex kit | Meso Scale Discovery | K15255D-1 | |
Commercial kit (ELISA) | TNF-alpha | Invitrogen | 88-7324-22 | |
Commercial kit (ELISA) | IL-6 | Invitrogen | 88-7064-22 | |
Commercial kit (ELISA) | IL-10 | Invitrogen | 88-7105-22 | |
Commercial kit (ELISA) | IL-1 beta | Invitrogen | 88-7013-86 | |
Commercial assay | Phagocytic assay | Cayman Chemical | 500290 | |
Other | Brewer thioglycollate | Sigma-Aldrich | B2551 | |
Other | DMEM/F12 GlutaMAX | ThermoFisher Scientific, Gibco | 10565018 | |
Other | CTAD tubes | BD Vacutainer | 367599 | |
Other | Peroxidase labelled streptavidin | Seracare | 71-00-38 | (1:200) |
Other | DAPI stain | Sigma-Aldrich | D9542 | (1:1000) |
Other | Ficoll | GE Healthcare | 17-5442-02 | |
Other | Myeloperoxidase assay | doi:10.1097/TP.0000000000000101 | ||
Strain, strain background (Mus musculus) | Wild-type mice | Harlan, Netherlands | C57BL/6J | |
Strain, strain background (Mus musculus) | MyD88/TRIF double-knockout mice | Mucosal Immunology, University of Bern | B6.129-MyD88tm1Aki x B6-Ticam1lps2 | |
Strain, strain background (Mus musculus) | Caspase-1/11 double-knockout mice | Mucosal Immunology, University of Bern | B6N.129S2-Casp1tm1Flv | |
Strain, strain background (Mus musculus) | MAC-CX43 knockout mice | Visceral Surgery, University of Bern | B6.129P2-Lyz2tm1(cre)Ifo/J x B6.129S7-Gja1tm1Dlg/J | Cell-specific conditional MAC-CX43 KO mouse model developed in our laboratory |
Strain, strain background (Mus musculus) | Lyz2-cre mice | Jackson Laboratory | B6.129P2-Lyz2tm1(cre)Ifo/J | |
Strain, strain background (Mus musculus) | Gja1 floxed mice | Jackson Laboratory | B6.129S7-Gja1tm1Dlg/J |
Anti-mouse antibodies used for Time-of-Flight Mass Cytometry.
(A) Diagnosis of human patients included in the study. (B) P2 receptors antagonists. (C) Visceral Surgery Murine Sepsis Score Sheet. (D) Anti-mouse antibodies used for flow cytometry (E) Anti-human antibodies used for flow cytometry (F) Anti-mouse antibodies used for Time-of-Flight Mass Cytometry
Full uncut western blot membranes.