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Pam2csk4

Manufactured by InvivoGen
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Pam2CSK4 is a synthetic bacterial lipopeptide that activates Toll-like receptor 2 (TLR2). It consists of a palmitic acid (Pam2) moiety coupled to the cysteine-serine-lysine (CSK) motif. Pam2CSK4 serves as a potent agonist for TLR2 and can be used to study TLR2-mediated immune responses in various research applications.

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Lab products found in correlation

Pam3csk4, by InvivoGen (51 mentions) Lipopolysaccharides (lps), by Merck Group (10 mentions) Imiquimod, by InvivoGen (6 mentions) Poly 1 c, by InvivoGen (5 mentions) Tnf α, by R&D Systems (5 mentions) Zymosan, by InvivoGen (4 mentions) Lipopolysaccharides (lps), by InvivoGen (4 mentions) Flagellin, by InvivoGen (4 mentions) Il 1β, by R&D Systems (3 mentions) Odn2006, by InvivoGen (3 mentions) Sb202190, by Merck Group (3 mentions) Brefeldin a, by Merck Group (2 mentions) Bay11 7082, by Merck Group (2 mentions) Pd98059, by Merck Group (2 mentions) Sp600125, by Merck Group (2 mentions) Poly 1 c hmw tlr 3 ligand, by InvivoGen (2 mentions) Anti gf of rsv, by US Biological (2 mentions) Ifn γ, by BioLegend (2 mentions) Rapamycin, by Selleck Chemicals (2 mentions) Ultrapure lps, by InvivoGen (2 mentions)

97 protocols using pam2csk4

1

Lipopolysaccharide and TLR Agonist Reagents

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Lipopolysaccharide (LPS; from E. coli 011:B4) and Brefeldin A (from Penicillium brefeldianum) were purchased from Sigma-Aldrich. TLR agonists Zymosan, Heat killed Listeria monocytogenes (HKLM), R848, low molecular weight Poly(I:C), purified Flagellin (from S. typhimurium), and synthetic bacterial lipoproteins Pam2CSK4 and Pam3CSK4, were purchased from InvivoGen (San Diego, CA).
Pattabiraman G., Palasiewicz K, & Ucker D.S. (2016). Toll-Like Receptor Function of Murine Macrophages, Probed by Cytokine Induction, Is Biphasic And Is Not Impaired Globally With Age. Mechanisms of ageing and development, 157, 44-59.
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2

Ovalbumin Immunization in Mice

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Mice were immunized with ovalbumin by intradermal injections at the base of the tail or i.v. injection into the lateral tail vein. Endotoxin free ovalbumin (EndoFit Ovalbumin, Invivogen) was used with a dose of 10 μg per injection in sterile PBS. Where noted, adjuvants were added to the immunogen, including Vaccigrade CpG-B (20 μg), Pam2CSK4 (10 μg), Pam3CSK4 (10 μg), high molecular weight Poly(I:C) (50ug) (all purchased from Invivogen), and MALP2 (1 μg, purchased from Enzo Life Sciences). For immunizations with live BCG, M. bovis BCG-Danish was acquired from the Statens Serum Institute (Copenhagen, Denmark) and cultured in Sauton medium (Difco Laboratories, BD Diagnostic Systems, Sparks, MD) with .05% Tween-80. Bacteria were grown from low passage number frozen stocks to mid-log phase and frozen in medium with 5% glycerol at −80°C in single use aliquots. For immunization, bacteria were thawed, washed and resuspended in PBS containing 0.05% Tween-80, and sonicated to obtain single-cell suspensions. Mice were vaccinated with 5 × 106 colony forming units (CFU) i.d. at the base of the tail or i.v. via lateral tail vein.
Johndrow C.T., Goldberg M.F., Johnson A.J., Ng T.W., Kunnath-Velayudhan S., Lauvau G., Gossel G.H., Kadolsky U.D., Yates A.J., Chan J., Jacobs WR J.r, & Porcelli S.A. (2018). Suppression of Th1 priming by TLR2 agonists during cutaneous immunization is mediated by recruited CCR2+ monocytes.. Journal of immunology (Baltimore, Md. : 1950), 201(12), 3604-3616.
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3

Macrophage Stimulation with TLR Ligands

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Raw264.7 (further Raw264) cells and J774 cells (both from ATCC) were cultured in DMEM containing 10% FBS (Thermo Fisher Scientific) with 4.5 and 1 g/l glucose, respectively. Thioglycolate-elicited macrophages were isolated from 8- to 14-week-old male C57BL/6 mice, as described previously [22 (link)]. The procedure had been reviewed and approved by the Local Animal Ethics Committee (permission No. 394/2017). For stimulation, Raw264 cells were overlaid with fresh DMEM/10% FBS supplemented with the following ligands: 10 or 100 ng/ml smooth LPS from Escherichia coli O111:B4 (List Biological Laboratories), 10 or 100 ng/ml N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine (Pam3CSK4), 10 or 100 ng/ml S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine (Pam2CSK4), 10 μg/ml polyinosinic-polycytidylic acid (poly(I:C), (the latter three from InvivoGen). Cells were stimulated for up to 6 h (5% CO2, 37 °C).
Matveichuk O.V., Ciesielska A., Hromada-Judycka A., Nowak N., Ben Amore I., Traczyk G, & Kwiatkowska K. (2024). Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14. Cellular and Molecular Life Sciences: CMLS, 81(1), 191.
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4

TLR Agonist Incubation Protocol

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For experiments using TLR agonists, cells were incubated with the agonists for the entire duration of the experiment. The sequence of CpG ODN 1668 (Integrated DNA Technologies) is: T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*T*G*C*T, with asterisks indicating phosphorothioate modifications. CpG ODN 1668 was used at a final concentration of 10μM. Pam2CSK4 (InvivoGen, Cat. tlrl-pm2s-1) was used at a final concentration of 100ng/mL. LPS (InvivoGen, Cat. tlrl-ekLPS) was used at a final concentration of 50ng/mL.
Nguyen B.N., Chávez-Arroyo A., Cheng M.I., Krasilnikov M., Louie A, & Portnoy D.A. (2020). TLR2 and endosomal TLR-mediated secretion of IL-10 and immune suppression in response to phagosome-confined Listeria monocytogenes. PLoS Pathogens, 16(7), e1008622.
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5

Immune Activation Signaling Pathways

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Pam3CSK4, Pam2CSK4, poly(I:C), flagellin, zymosan, and peptidoglycan (PGN) were purchased from InvivoGen (San Diego, CA, USA). Lipoteichoic acid (LTA) from S. aureus was prepared as previously described (29 (link)). Standard LPS (stdLPS) from Escherichia coli O111:B4 prepared by classical methods using phenol-water mixture extraction, and ultra-pure LPS (upLPS) prepared by successive enzymatic hydrolysis and phenol-triethylamine-deoxycholate extraction to remove other bacterial components from stdLPS were also obtained from InvivoGen. Fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI) medium and Dulbecco’s modified Eagle medium (DMEM) were obtained from HyClone (Logan, UT, USA). MAP kinase inhibitors, including PD98059 for ERK, SB202190 for p38, and SP600125 and JNK V inhibitor for JNK, and transcription factor inhibitors, such as BAY11-7082 for NF-κB, and mithramycin A for Sp1, were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals were from Sigma-Aldrich unless stated otherwise.
Im J., Baik J.E., Lee D., Park O.J., Park D.H., Yun C.H, & Han S.H. (2020). Bacterial Lipoproteins Induce BAFF Production via TLR2/MyD88/JNK Signaling Pathways in Dendritic Cells. Frontiers in Immunology, 11, 564699.
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6

Multiparametric Immune Cell Profiling

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Cetuximab (Erbitux) was purchased from Merck KGaA (Darmstadt, Hessen, Germany). Recombinant IL-15 was obtained from PeproTech (Rocky Hill, NJ, USA, cat. no. 200-15). The TLR ligands Pam2CSK4, Pam3CSK4 and flagellin were purchased from Invivogen (San Diego, CA, USA, cat. no. tlrl-pm2s-1, tlrl-pms and tlrl-bsfla) and lipopolysaccharide (LPS) from Sigma-Aldrich (Saint Louis, MO, USA, cat. no. L4391). Monensin (GolgiStop) was purchased from BD Biosciences (San Diego, CA, USA, cat. no. 554724). The monoclonal antibodies anti-CD69-BV412 (clone FN50, cat. no. 310930) and anti-CD107a-APC (clone H4A3, cat. no. 328620) were purchased from BioLegend (San Diego, CA, USA), while anti-CD56-PE (clone MY31, cat. no. 345810), anti-CD3-PerCP-Cy5.5 (clone SK7, cat. no. 332771), anti-PD-L1-BV786 (clone MIH1, cat. no. 563739), mIgG1-BV786 (clone X40, cat. no. 563330), anti-PD-L2-BV711 (clone MIH18, cat. no. 564258) and mIgG1-BV711 (clone X40, cat. no. 563044) were obtained from BD Biosciences. Goat polyclonal IgG F(ab)2 anti-hIgG-PE was purchased from Bio-Connect (Huissen, The Netherlands, cat. no. A59star97PE).
Gruijs M., Ganzevles S.H., Stigter-van Walsum M., van der Mast R., van Ostaijen-ten Dam M.M., Tuk C.W., Schilham M.W., Leemans C.R., Brakenhoff R.H., van Egmond M., van de Ven R, & Bakema J.E. (2021). NK Cell-Dependent Antibody-Mediated Immunotherapy Is Improved In Vitro and In Vivo When Combined with Agonists for Toll-like Receptor 2 in Head and Neck Cancer Models. International Journal of Molecular Sciences, 22(20), 11057.
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7

Reagents for Molecular and Cell Biology

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Cyanidin chloride (A18; CAS 528-58-5) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Pam2CSK4 (TLR2/TLR6 ligand) and poly(I:C)-HMW (TLR 3 ligand) were purchased from InvivoGen (San Diego, CA, USA). The mouse monoclonal antibody anti-gF of RSV was obtained from US Biological Life Sciences (Salem, MA, USA). The rabbit polyclonal antibody anti-Spike of SARS-CoV-2 was obtained from Sigma-Aldrich (St. Louis, MO, USA). Secondary goat anti-mouse FluoroLinkTM CyTM3 and goat anti-rabbit FluoroLinkTMCyTM2 antibodies were purchased from GE Healthcare (Chicago, IL, USA). DAPI was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Vicente J., Benedetti M., Martelliti P., Vázquez L., Gentilini M.V., Peñaranda Figueredo F.A., Nabaes Jodar M.S., Viegas M., Barquero A.A, & Bueno C.A. (2023). The Flavonoid Cyanidin Shows Immunomodulatory and Broad-Spectrum Antiviral Properties, Including SARS-CoV-2. Viruses, 15(4), 989.
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8

Cytokine Profiling of Monocyte Responses

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Whole blood was incubated with H37Rv (American Type Culture Collection [ATCC No. 25618, Rockville, MD]), LPS (E coli, strain 055.b5 lipopolysaccharide, Sigma Aldrich) or TLR agonists (ultra pure E coli LPS [tlrl-pelps]- TLR4, Pam2CSK4 [tlrl-pam2] – TLR2, Pam3CSK4 [tlrl-pms] – TLR2/1 and Poly(I:C) [tlrl-pic] – TLR 3, Invivogen, Nottingham, UK) for 18 h with addition of 1 μl Brefeldin A (Sigma Aldrich) after the initial two hours. Following lysis, fixation and permeabilization steps (FACSLysing Solution, Cytofix/Cytoperm and Perm/Wash Buffer, all BD Biosciences), cells were stained with FITC labelled anti-human CD14, PE labelled anti-human IL10, PerCP labelled anti-human HLA-DR and APC labelled anti-human TNFα for 15 min. Data was acquired using a BD FACSCalibur (BD Biosciences, Oxford, UK). Monocytes were identified on the basis of their forward scatter-side scatter characteristics. A minimum of 100 000 PBMC events were analysed. The percentage positivity in the unstimulated control sample was subtracted from the stimulated samples.
Waitt C.J., Banda P., Glennie S., Kampmann B., Squire S.B., Pirmohamed M, & Heyderman R.S. (2015). Monocyte unresponsiveness and impaired IL1β, TNFα and IL7 production are associated with a poor outcome in Malawian adults with pulmonary tuberculosis. BMC Infectious Diseases, 15, 513.
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9

Dendritic Cell Surface Marker Analysis

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After five days in culture, surface staining was performed on monocyte-derived dendritic cells (moDCs) for flow cytometry analysis. moDCs (0.8 × 106 cell/well) were then incubated with synthetic compounds in 12 wells at the reported concentrations. Stimulation with PAM2CSK4 1 µg mL−1 (Invivogen) was used as positive control. After 24 h, expression of all surface markers was estimated by using the following conjugated mAbs from Miltenyi Biotec: HLA-DR FITC, CD83 PE and CD86 APC, and analyzed by a flow cytometer (BD ACCURI, BD Bioscience, Milano, Italy) according to standard protocol.
Manzo E., Fioretto L., Gallo C., Ziaco M., Nuzzo G., D’Ippolito G., Borzacchiello A., Fabozzi A., De Palma R, & Fontana A. (2020). Preparation, Supramolecular Aggregation and Immunological Activity of the Bona Fide Vaccine Adjuvant Sulfavant S. Marine Drugs, 18(9), 451.
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10

Evaluating TLR responses to Lactobacillus

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Human embryonic kidney (HEK)-293 TLR reporter cell lines expressing human TLR1/2, TLR2/6, or TLR4, harboring pNIFTY, a NF-κB luciferase reporter construct (Invivogen, Toulouse, France) (Karczewski et al., 2010 (link)), were used. The HEK-293 reporter cell lines were seeded at 6 × 104 cells/well in 96-well plates and incubated overnight under standard culture conditions. Cells were then stimulated with late-stationary bacterial cultures of the L. plantarum NZ3400Cm, a L. plantarum WCFS1 derivative with a chromosomal integration of the cat cassette in a neutral chromosomal locus (Remus et al., 2012 (link)), and lgt deletion strain (NZ3565Cm) at a multiplicity of infection (MOI) of 1:10, HEK cell to bacteria. The TLR1/2 agonist Pam3CSK4 (5 μg/mL, Invivogen) and TLR2/6 agonist Pam2CSK4 (5 μg/mL, Invivogen) were used as positive controls and PBS served as the negative control.
Lee I.C., van Swam I.I., Boeren S., Vervoort J., Meijerink M., Taverne N., Starrenburg M., Bron P.A, & Kleerebezem M. (2020). Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1. Frontiers in Microbiology, 11, 1822.
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