Intrathecal injection into mice and analysis of the CSF were performed as described previously [7 (link),16 (link),19 (link)]. In detail, 40 μL of ligand solution (PCR studies after 12 h: 10 μg LPS, 10 μg Pam3CysSK4, 10 μg imiquimod, 10 μg CpG ODN; histologic studies after 72 h: 10 μg LPS, 40 μg Pam3CysSK4, 136 μg loxoribine, 10 μg CpG ODN, all obtained from Invivogen, or 10 μg HSP60 (Enzo Life Sciences) were injected intrathecally into 6- to 8-week-old male mice. Notably, the contamination with LPS of the HSP60 preparation as declared by the manufacturer was <50 EU/mg and ≤1.67 EU/mg, as determined by an independent laboratory specializing in endotoxin testing and published in previous work (Mikrobiologisches Labor, Münster, Germany; see also [18 (link)]). For real-time PCR analysis, brains were removed and cut along the sulcus medianus into two halves, which were separately snap-frozen in liquid nitrogen. For immunohistochemical studies, mice were perfused transcardially with isotonic-saline followed by 4% paraformaldehyde (PFA). Brains were removed, subjected to a row of 10%, 20%, and finally 30% sucrose in 0.1 M phosphate buffered saline (PBS) for cryoprotection, frozen in 2-methylbutan on dry ice, and stored at –80°C until sectioning.
Pam3cyssk4
Manufactured by InvivoGen
Sourced in United States
Pam3CysSK4 is a synthetic triacylated lipoprotein that acts as a Toll-like receptor 2 (TLR2) agonist. It is commonly used in research as a tool compound to stimulate the TLR2 signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharide lps, by InvivoGen (2 mentions)
Cpg odn, by InvivoGen (2 mentions)
Hsp60, by Enzo Life Sciences (2 mentions)
Imiquimod, by InvivoGen (2 mentions)
Anti cd4 apc cy7, by BioLegend (1 mentions)
Control igg, by R&D Systems (1 mentions)
Anti ifn γ pe cy7, by BioLegend (1 mentions)
Stat3 inhibitor stattic, by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Anti cd161 pe, by BD (1 mentions)
Anti human igg pe, by Jackson ImmunoResearch (1 mentions)
Anti cd3 cd28 microbeads, by Miltenyi Biotec (1 mentions)
Anti il 17 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd45ra fitc, by BD (1 mentions)
Anti cd45ro pe, by BD (1 mentions)
Anti il 23p19, by R&D Systems (1 mentions)
Golgiplug, by BD (1 mentions)
Hyclone fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Resiquimod r848, by InvivoGen (1 mentions)
7 protocols using pam3cyssk4
1
Intrathecal Injection and CSF Analysis in Mice
2
Comprehensive Immune Cell Profiling
3
Bone Marrow Derived Macrophage Stimulation
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Bone marrow derived macrophages (BMDM) from wild-type and knockout mice were cultured in RPMI with 10% fetal bovine serum (Thermo Scientific HyClone Fetal Bovine Serum (U.S.), Defined SH3007003HI Heat inactivated) and 20% L929 supernatants [49] (link) and were allowed to mature into macrophages over 7 days. Cells were seeded into 24-well plates at 2×105 cells/well (ELISA assays) or 6-well plates at 1×106 cells/well (Western blot analysis) and stimulated with bacteria at indicated MOI overnight. Cells stimulated with LPS from E. coli OIII:B4 (InvivoGen) or Pam3CysSk4 (InvivoGen) served as controls.
4
Stimulation and Cytokine Measurement
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Blood cell stimulation employed flat-bottom 6-well plates (TPP, Switzerland). Per well, 5 × 106 cells were cultured in 3 mL Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies).
The synthetic TLR ligands Pam3Cys-SK4 (10 μg/mL), polyinosine–polycytodylic acid (polyI:C) (10 μg/mL), lipopolysaccharide (LPS) (10 μg/mL) and resiquimod (R848) (10 μg/mL), all from InvivoGen, were used for stimulation of cells over 18 h. Likewise the mitogens phorbol 12-myristate 13-acetate (PMA) (200 ng/mL), ionomycin (1 μg/mL) and concanavalin A (10 μg/mL), all from Sigma-Aldrich, were used for stimulation of cells over 18 h.
Stimulation with M. bovis (live or heat-inactivated) was done at a multiplicity of infection (MOI) of 0.1, based on preliminary experiments, for 18 h.
After 14 h, 50–100 μL of cell culture supernatant was collected and frozen for further cytokine secretion measurement (multiplex immunoassay). As soon after, Brefeldin A (10 μg/mL) (ThermoFisher) was added to the medium to block cytokine secretion; incubation was extended for another 4 h, to allow the de novo cytokine synthesis measurement (flow cytometry intracellular staining).
The synthetic TLR ligands Pam3Cys-SK4 (10 μg/mL), polyinosine–polycytodylic acid (polyI:C) (10 μg/mL), lipopolysaccharide (LPS) (10 μg/mL) and resiquimod (R848) (10 μg/mL), all from InvivoGen, were used for stimulation of cells over 18 h. Likewise the mitogens phorbol 12-myristate 13-acetate (PMA) (200 ng/mL), ionomycin (1 μg/mL) and concanavalin A (10 μg/mL), all from Sigma-Aldrich, were used for stimulation of cells over 18 h.
Stimulation with M. bovis (live or heat-inactivated) was done at a multiplicity of infection (MOI) of 0.1, based on preliminary experiments, for 18 h.
After 14 h, 50–100 μL of cell culture supernatant was collected and frozen for further cytokine secretion measurement (multiplex immunoassay). As soon after, Brefeldin A (10 μg/mL) (ThermoFisher) was added to the medium to block cytokine secretion; incubation was extended for another 4 h, to allow the de novo cytokine synthesis measurement (flow cytometry intracellular staining).
5
Microglial and Neuronal Responses to TLR Ligands
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Lipopolysaccharide (LPS), Pam3CysSK4, loxoribine, imiquimod, and CpG ODN were obtained from Invivogen, San Diego, USA. HSP60 was obtained as low-endotoxin charge from Enzo Life Sciences, Lörrach, Germany. Dose response studies with the different ligands were performed in microglial and neuronal cultures analyzed by TNF-α ELISA (Additional file 1 : Figure S1A) and by quantification of NeuN+ cells per field (Additional file 1 : Figure S1B), respectively. The concentrations of the respective ligands that induce maximum responses were in part published in previous work [7 (link),18 (link),19 (link)] and were used in this study.
6
Alveolar Epithelial Cell Stimulation
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AEC were seeded in 12-well polystyrene tissue culture-treated plates (Corning Costar) at cell densities of 4 x 104 cells/cm2. At confluence, AEC were starved in starvation medium (DMEM or EMEM containing 0.5% heat-inactivated FBS media, penicillin streptomycin) for 16 h prior to stimulation. After starvation, PAF and/or SAF were added at the indicated concentrations (2.5 to 10% v/v). AEC were then incubated at 37°C with 5% CO2 for 6 h unless otherwise specified. For stimulation with TLR1/2, TLR4 and TLR5 agonists, Pam3CysSK4, E. coli LPS, or S. typhimurium flagellin (Invivogen) were used at the indicated concentrations. For stimulation of the EGFR-dependent pathway, Human Epidermal Growth Factor (hEGF) (Roche Diagnostics) was used at the indicated concentration. AEC were stimulated with LB broth medium as negative control. After stimulation, the conditioned AEC supernatants were collected, centrifuged at 13,000 g for 10 min to pellet cell debris, and stored at -20°C until further analysis.
7
Microglial Activation and γδ T Cell Interaction
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Microglia were plated at 30x103/96-well in 200 μl DMEM supplemented with 10% heat inactivated FCS, 1% penicillin/streptomycin and left to adhere overnight. After removal of 100 μl of media cells were stimulated with the TLR ligands Pam3CysSK4 (100ng/ml), imiquimod (10μg/ml) (all from InvivoGen, Toulouse, France), LPS (100ng/ml, Enzo Life Sciences GmbH, Lörrach, Germany), CpG 1668 (1μM, TIB MolBiol, Berlin, Germany) for 24 h. Subsequently, conditioned microglial supernatants were transferred to naïve γδ T cells (30x103/96-well in 100 μl complete RPMI), or naïve γδ T cells were co-cultured with stimulated microglia at a 1:1 ratio. After indicated time points cells were collected for flow cytometry and supernatants were recovered for ELISA or multiplex analysis of cytokines, as indicated. TLR stimulation of bone marrow-derived macrophages was carried out likewise. For neutralization of IL-1β and IL-23, conditioned microglial supernatants were pre-incubated for 1 h at 4°C with 10 μg/ml anti-IL-1β (clone B122), anti-IL-23 (p19, clone MMp19B2) or respective isotype controls (all obtained from BioLegend, San Diego, USA) before supernatants were used for incubation of naïve γδ T cells.
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