Monocyte cytokine production was analyzed using freshly isolated monocytes from 8 healthy Kenyan children, 10 healthy Kenyan adults, and 10 healthy US adults, as previously described [35 (link)]. Monocytes were isolated from whole blood via negative selection with the RosetteSep Human Monocyte Enrichment Cocktail (Stemcell Technologies, 15,068). Cells were suspended in culture medium (RPMI-1640 supplemented with 2 mM L-glutamine, 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, and 0.05 mM 2-ME) and placed in 96-well polypropylene plates at 5 × 104 cells per well (concentration 5 × 105 cells/ml). Cells were stimulated with 10 ng/ml LPS (Sigma-Aldrich) and 100 ng/ml Pam3CSK4 (P3C) (Invivogen) and compared with a media-alone control; each condition was performed in duplicate. Cells were cultured for 18 h at 37 °C, in 5% CO2, on an orbital shaker. Supernatants were harvested and stored at − 80 °C. A multiplex magnetic bead–based immunoassay was used to measure concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF (EMD Millipore) in the culture supernatants immediately after initial thawing.
Pam3csk4 p3c
Manufactured by InvivoGen
Sourced in Hong Kong, France, United States
Pam3CSK4 (P3C) is a synthetic triacylated lipoprotein that serves as a Toll-like receptor 1 (TLR1)/TLR2 agonist. It is a well-characterized and commonly used tool in immunology research.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Mtt assay, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Tumor necrosis factor (tnf), by Merck Group (1 mentions)
Rosettesep human monocyte enrichment cocktail, by STEMCELL (1 mentions)
Il 12p70, by Merck Group (1 mentions)
Il 1β, by Merck Group (1 mentions)
Il 10, by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Bay11 7085, by Santa Cruz Biotechnology (1 mentions)
U0126, by InvivoGen (1 mentions)
Cyt387, by Selleck Chemicals (1 mentions)
Pam2cgdpkhpksf, by InvivoGen (1 mentions)
Sp600125, by Selleck Chemicals (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
Kdo2 lipida kla, by Avanti Polar Lipids (1 mentions)
Neomycin sulfate, by Solarbio (1 mentions)
Sodium butyrate, by Solarbio (1 mentions)
Metronidazole, by Solarbio (1 mentions)
Ampicillin, by Solarbio (1 mentions)
10 protocols using pam3csk4 p3c
1
Monocyte Cytokine Production in Kenyan and US Populations
2
Toll-like Receptor Signaling in GC Cells
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GC cells were grown in 6‐well plates in triplicate. After overnight starvation, cells were treated with phosphate‐buffered saline (PBS control), Pam3CSK4 (P3C, InvivoGen), Lipopolysaccharides from Escherichia coli O111:B4 (LPS, Sigma) or FSL‐1 (Pam2CGDPKHPKSF, InvivoGen) at the indicated concentrations after a time course. For transfection experiments, GC cells at 50–60% confluence in 6‐well plates were transiently transfected with 10 nM nontarget control or siRNA targeting SOD2 gene (Ambion) using Lipofectamine 3000 (ThermoFisher). For signaling pathway analysis, cells were pre‐treated for 1 h with either DMSO vehicle; the JAK inhibitor, CYT387 (Selleck); the JNK inhibitor, SP600125 (Selleck); the ERK inhibitor, U0126 (InvivoGen); the PI3K inhibitor, Wortmannin (Sigma); the NF‐κB inhibitor, BAY11‐7085 (Santa Cruz Biotechnology); or the P38 inhibitor, SB203580 (Selleck) at the indicated concentrations prior to stimulation with P3C or PBS. Cell viability assay in response to TLR agonists’ treatment was also conducted using the MTT assay (Invitrogen) in 96‐well plate after the manufacturer's instructions.
3
Bacterial Strain Preparation for Macrophage Stimulation
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Bacterial strains used were as follows: Pseudomonas aeruginosa (PA01), Salmonella enterica Typhimurium (14028 s), Escherichia coli (K12 MG1655), Staphylococcus aureus (FDA209), and Listeria monocytogenes (ΔactA strain DPL1942; Brundage et al., 1993 (link)). Bacteria were grown up overnight in either Luria-Bertani Miller broth (Pseudomonas, Salmonella, and E. coli) or trypticase soy broth (Staphylococcus and Listeria) shaking at 37° C. Absorbance at 600 nm was measured to determine bacterial concentration. Bacteria were washed 2x in PBS, then heat-killed at 60–95° C (depending on bacteria) for 1 hr. Heat-killed bacteria were stored at 4° C. Bacteria were diluted to 1 × 109 CFU/mL in PBS, representing a 10X solution (1X being equivalent to MOI of 100), and Kdo2-LipidA (KLA, Avanti Polar Lipids) and Pam3CSK4 (P3C, Invivogen) were diluted in complete DMEM, before addition to BMDM.
4
Immune Modulation via Pharmacological Agents
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Ampicillin, neomycin sulfate, metronidazole, vancomycin, vitamin A, and sodium butyrate were purchased from Solarbio life science (Beijing, China). TLR2 ligands Pam3CSK4 (P3C) was purchased from InvivoGen (Hongkong, China). Recombinant chicken GM-CSF protein (rGM-CSF) and recombinant chicken IL-17 protein (rIL-17) were purchased from Abcam (Cambridge, MA, UK).
5
Microbial Particle Phagocytosis Assay
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pHrodo red E. coli and S. aureus as well as AF488-conjugated E. coli bioparticles were purchased from Thermo Fisher Scientific. Ultrapure 0111:B4, K12 LPS from E. coli, poly I:C, imidazoquinoline compound R848 (Resiquimod), thiazoloquinoline compound CL075, and synthetic diacylated lipoproteins FSL-1 (Pam2CGDPKHPKSF) and Pam3CSK4 (P3C) were from InvivoGen. Ultrapure K12 LPS or 0111:B4 LPS (InvivoGen) were used at concentrations of 100 ng/ml. E. coli bioparticles were reconstituted in 2 ml PBS, and 50 µl/well (1.5 × 107 particles) in 1 ml of media was used for cells in six-well plates (Nunc) or 35-mm glass-bottomed tissue cell dishes (MatTek Corporation), and 15 µl/well (0.45 × 107 particles) in 0.5 ml of media were used for 24-well plates (Nunc). The pan-Akt inhibitor MK2206 (1032350-13-2; Axon Medchem) and the TBK1-IKKε inhibitor MRT67307 (from P. Cohen, University of Dundee, Dundee, Scotland, UK; Clark et al., 2011 (link)) were diluted in DMSO at a concentration of 20 mM and stored at −80°C, and working solutions were prepared in cell culture media immediately before use.
6
Inflammasome Activation by NLRC4
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NLRC4-specific stimulation was performed by priming with TLR1/2 agonist Pam3CSK4 (P3C) at 100 ng/µL (InvivoGen, San Diego, CA) and simultaneous retroviral transduction with pMXsIG_PrgI_GFP in presence of polybrene (8 μg/ml) as previously described [6 (link)]. After 24 h, supernatants were collected for quantification of IL-1β, IL-18, and IL-8 cytokine levels by ELISA and cell death was analyzed by flow cytometry following propidium iodide (PI) staining (1 µg/ml, Sigma-Aldrich, St. Louis, MO) of cells. The NLRP3 inflammasome was stimulated with 10 μM Nigericin (InvivoGen, San Diego, CA) for 1 h after 3 h priming with P3C (100 ng/µL).
7
Diluting TLR2 Agonists: Avoiding Activity Loss
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TLR2 agonists Pam3CSK4 (P3C) and peptidoglycan (PGN) were purchased from Invivogen (Toulouse, France). The lipopetides were resuspended in endotoxin free water and stored at -20 °C as aliquots of 200 μg/ml for further dilution. The bioactivity of lipopeptide TLR2 agonists is highly dependent on the dilution protocol employed. There might be a loss of activity of these lipopeptides when diluted in protein- and serum-free buffers. This is due to the formation of large aggregates of lipopeptides when not solubilized properly.24 (link)
8
LPS, P2C, and P3C Reagent Acquisition
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Lipopolysaccharide A (LPS) (Escherichia coli 0111:B4) was obtained from Sigma‐Aldrich (St Louis, MO, USA). Purified Pam2CSK4 (P2C) and Pam3CSK4 (P3C) were obtained from InvivoGen (San Diego, CA, USA). In some experiments, P3C was obtained from Bachem Americas, Inc. (Torrance, CA, USA).
9
Bone Marrow-Derived Macrophage Priming
10
TLR Ligands Viability Assay
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GC cell lines were grown in 12-well plates in triplicate (1 × 10 5 cells/well). After serum-free starvation overnight, cells were treated with phosphate-buffered saline (PBS control), Pam3CSK4 (P3C, InvivoGen) 10 µg/ml, Lipopolysaccharides from Escherichia coli O111:B4 (LPS, Sigma) 10 ng/ml, FSL-1 (Pam2CGDPKHPKSF, InvivoGen) 1 µg/ml, Heat Killed Listeria monocytogenes (HKLM, InvivoGen) 10 7 -10 8 cells/ml, ODN2006 (ODN7909, Stimulatory CpG ODN, Class B, InvivoGen) 2 µM for 24 h. Cell viability assay in response to TLR ligands for cell number normalization was also conducted using the MTT assay (Invitrogen) in 96-well plate following the manufacturer's instructions.
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