Pam3csk4 pam3

Manufactured by InvivoGen

Sourced in United States, France

Pam3CSK4 (Pam3) is a synthetic triacylated lipopeptide that mimics the acylated amino terminus of bacterial lipoproteins. It is a Toll-like receptor 1/2 (TLR1/2) agonist that can be used in cell culture and research applications.

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Pam3CSK4 (641 citations)

6 protocols using pam3csk4 pam3

Heat Stress Response in Macrophages

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Primary mouse BMDMs, primary human macrophages, primary human monocytes, THP-1 cells, RAW264.7 cells, and L929 cells were stimulated with the following ligands where indicated unless otherwise noted: 100 ng/ml LPS (InvivoGen, tlrl-3peLPS), 1 μg/ml Pam3CSK4 (Pam3; InvivoGen, tlrl-pms), 1 μg/ml poly(I:C) (InvivoGen, tlrl-picw), 1 μg/ml Imiquimod (InvivoGen, tlrl-imqs), 1 μg/ml CpG (InvivoGen, tlrl-1585), 25 μM zVAD, 30 μM Nec-1S, 1 μM MCC950, 5 μM glycine (mouse BMDMs) and 50 μM glycine (human macrophages) (Sigma-Aldrich, G7126) for 2 h. Then, cells were placed in an incubator at 43 °C and 5% CO₂ for indicated times to induce heat stress (HS). After HS exposure, cells were incubated at 37 °C for the indicated times. Cell lysates and supernatants were collected at the indicated timepoints after HS for western blot and immunoprecipitation.
For bacterial infection, the BMDMs were infected separately with the following bacteria: 5 MOI of E. coli or 5 MOI of C. rodentium for 3 h. Then, cells were washed three times with PBS, and fresh media supplemented with 50 μg/ml gentamicin (Thermo Fisher Scientific, 15750-060) was added to kill the extracellular bacteria. Then the cells were subjected to HS as described above.

Han J.H., Karki R., Malireddi R.K., Mall R., Sarkar R., Sharma B.R., Klein J., Berns H., Pisharath H., Pruett-Miller S.M., Bae S.J, & Kanneganti T.D. (2024). NINJ1 mediates inflammatory cell death, PANoptosis, and lethality during infection conditions and heat stress. Nature Communications, 15, 1739.

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Immune Modulation Reagents and Assays

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The following reagents, antibodies and kits were used in the present study: recombinant mouse GM-CSF and IL-4 (R&D Systems, Minneapolis, MN); recombinant human interleukin 2 (IL-2) (Chiron Corp., Emeryville, CA); high purity TLR4 ligand (with less than 3% impurities), Escherichia coli serotype 055:B5 lipopolysaccharide (LPS) (part No 7193, lot No GL1457; Lonza, Walkersville, MD); synthetic TLR1/2 agonist Pam₃CSK4 (Pam₃, InvivoGen, San Diego, CA); phycoerythrin (PE)-conjugated rat anti-mouse TNF, CD14 and TLR4 monoclonal antibodies (BD-Pharmingen, CA, USA); unconjugated rat anti-mouse TNF (XT22, Pierce-Endogen, Rockford, IL), human TNFR2-Fc fusion protein (ENBREL, etanercept; Amgen, Thousand Oaks, CA), hamster anti-mouse TNFR1 and TNFR2 (BD-Pharmingen) and isotype control monoclonal antibodies (BD-Pharmingen); dominant negative TNF constructs (DNTNF1, XPro1595; and DNTNF2, XENP550; Xencor, Monrovia, CA); Limulus Amebocyte Lysate (LAL) Chromogenic Assay kit (Thermo Fisher Scientific Inc, Pittsburgh, PA); mouse Quantikine IFN-γ enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems); and mouse TNF DuoSet ELISA kits (R&D Systems).

Sobo-Vujanovic A., Munich S, & Vujanovic N.L. (2014). Dendritic-cell exosomes cross-present Toll-like receptor-ligands and activate bystander dendritic cells. Cellular immunology, 289(0), 119-127.

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Endothelial Barrier Disruption by Monocyte Transmigration

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Endothelial cell monolayer disruption (transmigration) by monocytes +/− LPS and TLR2 ligand stimulation was measured using the 9600Z Electrical Cell-Substrate Impedance Sensing (ECIS) system (Applied BioPhysics, Troy, NY, USA). A 96-well plate containing 20 gold film electrodes per well (ibidi) was coated with 1 mg/mL neutralized rat tail collagen type I (Thermo Fisher Scientific) for 10 min at room temperature and 2 µg/mL bovine plasma fibronectin (Thermo Fisher Scientific) for 45 min at 37 °C and 5% CO₂. Wells were then inoculated with LECs at a seeding density of 30,000 cells per well in 100 µL huMEC complete media at 37 °C and 5% CO₂. The run was performed under a single frequency of 4000 Hz and continuously monitored every 60 s. Impedance (Z) was determined by Ohm’s law: Z = V/I. After 20 h, when an endothelial monolayer was reached, KO and WT THP-1 cells with and without stimulants were added at a density of 100,000 cells per well in 50 µL huMEC complete media. For stimulation, 100 ng/mL LPS (Thermo Fisher Scientific) or 300 ng/mL of either TLR2-specific ligand Pam2CSK4 (Pam2) or Pam3CSK4 (Pam3) (both InvivoGen) was used. Endothelial monolayer disruption was observed for 10 h.

Colleselli K., Ebeyer-Masotta M., Neuditschko B., Stierschneider A., Pollhammer C., Potocnjak M., Hundsberger H., Herzog F, & Wiesner C. (2023). Beyond Pattern Recognition: TLR2 Promotes Chemotaxis, Cell Adhesion, and Migration in THP-1 Cells. Cells, 12(10), 1425.

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Immune Cell Culture Reagents

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Cell culture media RPMI 1640 GlutaMAX™ and DMEM GlutaMAX™, PBS, non-essential amino acids (NEAA), sodium pyruvate and antibiotics [Penicillin–Streptomycin (Pen–Strep™), Gentamicin™, Normocin™, and Zeocin™] were purchased from GIBCO (Life Technologies). Fetal bovine serum (FBS) was provided by Eurobio, France. Zymosan (Zym), particulate and dispersible whole glucan particle (WGPd, Biothera) and soluble whole glucan particle compounds (WGPs, Biothera) and curdlan (Curd), a linear β1,3-glucan extracted from Alcaligenes faecalis, synthetic triacylated lipoprotein Pam3CSK4 (Pam3) and ultraPure LPS from Escherichia coli O111:B4 were purchased from InvivoGen (France). BG compounds of interest were extracted from the same strain of S. cerevisiae owned by Phileo-Lesaffre Animal Care. Their composition was already described in a recent study (23 (link)). The previous name given to the crude compounds BG15 was replaced in this study by ScCW for better understanding and more convenience.

Walachowski S., Tabouret G., Fabre M, & Foucras G. (2017). Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response. Frontiers in Immunology, 8, 1089.

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Monocyte Activation in Systemic Lupus Erythematosus

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Blood was obtained from SLE patients and healthy volunteers after obtaining IRB-approved written consent (NIDDK/NIAMS IRB, Bethesda, MD). PBMC were isolated by density centrifugation over Histopaque-1077 (Millipore Sigma, Merck) at 400 g for 30 min. Monocytes were sorted using human CD14 Microbeads (MACS, Miltenyi Biotec) according to the manufacturers’ protocol. Briefly, 20 μl of CD14 microbeads were added to 10⁷ cells suspended in 80 μl of MACS buffer. After 15 min incubation in the dark at 4° C cells were washed, resuspended in 500 μl of MACS buffer and loaded onto magnetized LS columns. The columns were extensively washed and CD14⁺ cells collected by removing the magnet and flushing with 5 ml of MACS buffer.
10⁵ purified monocytes/well were seeded into 96-well flat bottom plates (Corning) and stimulated for 5 days at 37°C with 1 μg/ml PAM3CSK4 (PAM3) (Invivogen), 250 ng/ml MCSF (Miltenyi Biotec), 250 ng/ml IFNγ (Miltenyi Biotec) or 1 μl /ml R848 (Invivogen). Previous studies established that these were the optimally stimulatory concentrations of each agent ([14 (link),15 (link)] and data not shown). Cells were maintained in medium consisting of RPMI 1640 (Lonza) supplemented with 2% heat-inactivated fetal calf serum 25 nmol/L HEPES, 1 mmol/L sodium pyruvate, non-essential amino acids (NEAAs) and 0.0035% 2-mercaptoethanol (2-ME).

Horuluoglu B., Bayik D., Kayraklioglu N., Goguet E., Kaplan M.J, & Klinman D.M. (2019). PAM3 supports the generation of M2-like macrophages from lupus patient monocytes and improves disease outcome in murine lupus. Journal of autoimmunity, 99, 24-32.

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Inflammasome Activation Protocols

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Cells were seeded at 1x10⁶ cells/ml and incubated overnight at 37°C, 5% CO₂. Adherent cells were primed with either ultrapure E.coli lipopolysaccharide (LPS) (InvivoGen) at a final concentration of 200 ng/ml for 3 hours or Pam3CSK4 (Pam3) (InvivoGen) at a final concentration of 10 μg/ml for 4 hours at 37°C, 5% CO₂. Cells were stimulated with nigericin (Sigma-Aldrich) at a final concentration of 20 μM to 200 μM for 1 hour or at a final concentration of 10 μM for 24 hours. For non-canonical inflammasome activation, cells were incubated with ultrapure LPS at 5 μg/ml to 20 μg/ml final concentration in the presence of FuGENE HD transfection reagent (Promega) for 16 hours at 37°C, 5% CO₂. LPS and transfection reagent were pre-incubated for 15 minutes at room temperature prior to addition to the cells. For pharmacological inhibition of caspase-8, cells were pre-incubated with the caspase-8 Z-IETD-FMK inhibitor (R&D Systems) at a final concentration of 10 μM and and re-supplemented every time the cell culture medium was changed.

Digby Z., Tourlomousis P., Rooney J., Boyle J.P., Bibo-Verdugo B., Pickering R.J., Webster S.J., Monie T.P., Hopkins L.J., Kayagaki N., Salvesen G.S., Warming S., Weinert L, & Bryant C.E. (2021). Evolutionary loss of inflammasomes in the Carnivora and implications for the carriage of zoonotic infections. Cell Reports, 36(8), 109614.

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