Pam3csk4

Manufactured by Merck Group

Sourced in United States, Germany

Pam3CSK4 is a synthetic lipopeptide that acts as a Toll-like receptor 1/2 (TLR1/2) agonist. It is commonly used as a research tool in immunology and microbiology studies.

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Lab products found in correlation

52 protocols using pam3csk4

NAD+ Modulation of Inflammasome Activation

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After 8 days of culture the medium was replaced by 40 ng/ml GM-CSF containing 100 µmol NAD⁺ culture medium. For 2 following days NAD⁺ was added daily until stimulation. To induce canonical and non-canonical inflammasome activation in murine macrophages, NAD⁺-treated and control BMDMs were cultured overnight in a 24-well plate at 1×10⁶ cells/ml and afterward primed with 1 µg/ml Pam3CSK4 or 1 µg/ml LPS O111:B4 (Sigma) for 5–6 hr. Primed BMDMs were then stimulated for 16 hr with either 5 mmol ATP (canonical inflammasome stimulation) or 2 µg/ml LPS O111:B4 and 20 µg/ml CTB (Sigma) to allow LPS entry (non-canonical inflammasome stimulation) where indicated. To test the effect of NAD⁺ on type 1 IFN and STAT1 signaling, BMDMs were cultured overnight in a 24-well plate at 1×10⁶ cells/ml and afterward primed with 1 µg/ml Pam3CSK4 or 1 µg/ml LPS O111:B4 (Sigma) for 5–6 hr. Primed BMDMs were then stimulated for 16 hr 2 µg/ml LPS O111:B4, 20 µg/ml CTB, and U/ml recombinant IFN-β.

Iske J., El Fatimy R., Nian Y., Ghouzlani A., Eskandari S.K., Cetina Biefer H.R., Vasudevan A, & Elkhal A. (2024). NAD+ prevents septic shock-induced death by non-canonical inflammasome blockade and IL-10 cytokine production in macrophages. eLife, 12, RP88686.

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Modulating Cell Line Responses to Inflammatory Stimuli

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The LX-2 and HepG2 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in DMEM (BI, Israel) supplemented with 10% fetal bovine serum (FBS, BI) and 1% penicillin/streptomycin at 37°C with 5% CO 2 . In the experimental groups, LX-2 cells were exposed to ST2825 (10 µM) (MedChemExpress, USA) for 2 h, and then were challenged with TLR1/2 agonist (Pam3CSK4, 100 ng/mL), TLR4 agonist (LPS, 1 mg/mL) and palmitic acid (400 µM) (Sigma, USA) respectively for 24 h. In the control groups, LX-2 cells were directly challenged with TLR1/2 agonist (Pam3CSK4, 100 ng/mL), TLR4 agonist (LPS, 1 mg/mL) and palmitic acid (400 µM) (Sigma, USA) respectively for 24 h. To induce excessive lipid accumulation, HepG2 cells were cultured in DMEM medium with 20 µM AKT inhibitor (CSNpharm, USA) for 24h, then 400 µM palmitic acid and 200 ng/mL recombinant protein OPN (Cloud-Clone Corp, China) for 24h [32] .

Li Y., Wei M., Yuan Q., Liu Y., Tian T., Hou L, & Zhang J. (2022). MyD88 in hepatic stellate cells promotes the development of alcoholic fatty liver via the AKT pathway. Journal of molecular medicine (Berlin, Germany), 100(7).

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Regulation of Aquaporin-3 in Murine Keratinocytes

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Mouse keratinocytes were isolated from the skin of newborn ICR CD1 mice and cultured as described previously [63 (link)], according to protocols approved by the Institutional Care and Use Committees of Augusta University (Protocol #2017-0915) and the Charlie Norwood VA Medical Center (Protocol #22-04-135). Keratinocytes were treated with 0, 2.0 µg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA), or 2.5 µg/mL Pam₃CSK₄ (EMD Millipore Corporation, Burlington, MA, USA) in the presence or absence of 1 µM SAHA (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO₂ for 24 h; or with 0, 50, or 100 µg/mL AGE-BSA (Sigma-Aldrich or BioVision Incorporated, Milpitas, CA, USA), in the presence or absence of 1 or 2 µM SAHA (based on our previous studies [40 (link)]) at 37 °C with 5% CO₂ for 24, 48, and 72 h. Doses of AGEs were selected based on previous literature [54 (link),64 ,65 (link),66 (link)]. Cells were then harvested for RT-qPCR or Western analysis. Cells treated with SAHA were analyzed separately, to determine whether TLR2/TLR4 activation or AGEs altered AQP3 expression and/or function under basal conditions and settings analogous to diabetes (with enhanced protein acetylation).

Luo Y., Uaratanawong R., Choudhary V., Hardin M., Zhang C., Melnyk S., Chen X, & Bollag W.B. (2023). Advanced Glycation End Products and Activation of Toll-like Receptor-2 and -4 Induced Changes in Aquaporin-3 Expression in Mouse Keratinocytes. International Journal of Molecular Sciences, 24(2), 1376.

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Investigating HIV Infection and Immune Response in Memory CD4 T Cells

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Most experiments involved culture of uninfected or HIV-infected memory CD4 T cells in IL2 (20ng/ml) medium for up to 1 week, followed by data collection. Cells were cultured in 48-well plates (5x10⁵ cells in 1ml medium) with appropriate reagents. The HIV antiviral inhibitor, Saquinavir, was acquired from NIHAIDS Reagent Program and used at 1–10μg/ml. The GrzB/caspase inhibitor, Z-IETD-FMK (Enzo Life Sciences, Farmingdale, NY USA), was used at 10μM. Experiments involving TLR stimulation of memory CD4 T cells utilized synthetic TLR ligands LPS (Sigma, St. Louis, MO USA), Pam3CSK4 (EMD Millipore, Billerica, MA USA), peptidoglycan, Poly(I:C), and Poly(U) (InvivoGen, San Diego, CA USA), and were used at 2–25μg/ml +/− CD3 activation (clone UCHT1 coated at 0.1μg/ml).

Couturier J., Hutchison A.T., Medina M.A., Gingaras C., Urvil P., Yu X., Nguyen C., Mahale P., Lin L., Kozinetz C.A., Schmitz J.E., Kimata J.T., Savidge T.C, & Lewis D.E. (2014). HIV replication in conjunction with granzyme B production by CCR5+ memory CD4 T cells: implications for bystander cell and tissue pathologies. Virology, 0, 175-188.

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TLR Agonists Enhance Cytokine Secretion in AML

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The following TLR-agonists (InvivoGen; San Diego, CA, USA, if not stated otherwise) were used at following concentrations: (i) 10 ng/mL of the TLR1/2 heterodimer agonist Pam3CSK4, a synthetic triacetylated lipopeptide, (ii) 10 ng/mL of the TLR4 agonist LPS isolated from Escherichia coli 0111:B4 (Sigma Aldrich; St. Louis, MO, USA), (iii) 100 ng/mL of the TLR5 agonist flagellin isolated from Salmonella typhimurium, and (iv) 100 ng/mL of the dual TLR7/8 agonist resiquimod (R848). These compounds were tested on ten unselected AML patients at concentrations of 0.01, 0.1 and 1.0 µg/mL (Pam3CSK4, LPS and flagellin) or respectively of 0.1, 1.0 and 10 µg/mL (R848). At the selected concentrations, the agonists significantly increased the AML blasts’ secretion of the cytokines CCL2, CCL3, CCL4, CXCL8 and IL-6, while the proliferation capacity of the cells was not compromised (Figure S3).

Brenner A.K, & Bruserud Ø. (2019). Functional Toll-Like Receptors (TLRs) Are Expressed by a Majority of Primary Human Acute Myeloid Leukemia Cells and Inducibility of the TLR Signaling Pathway Is Associated with a More Favorable Phenotype. Cancers, 11(7), 973.

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Pectin-based Inflammatory Bowel Assays

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Citrus pectin (derived from lemon and lime peels) and orange pectin were kindly provided by CP Kelco ApS (Lille Skensved, Denmark). Dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzoic sulfonic acid (TNBS) and were obtained from Wako Pure Chemical Industries (Osaka, Japan). Lipopolysaccharide (LPS, Escherichia coli O111: B4) and Pam3CSK4 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Ishisono K., Mano T., Yabe T, & Kitaguchi K. (2019). Dietary Fiber Pectin Ameliorates Experimental Colitis in a Neutral Sugar Side Chain-Dependent Manner. Frontiers in Immunology, 10, 2979.

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Comparative LPS Bioactivity Analysis

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The Pg-LPS as purchased from Wako (Tokyo, Japan) was used in the in vivo experiments. Other Pg-LPS were tested for a comparative examination of the Wako Pg-LPS activity to TLR2 and TLR4: LPS obtained from P. gingivalis was cultured with hemin at high and low concentrations (Darveau laboratory) because of the requirement of hemin as an iron source for the growth of P. gingivalis, Pg-LPS purchased from Invivogen (San Diego, California, USA), and LPS consisting of tetra-acylated lipid A structures with low induction of the host response (Pg-LPS1435, Darveau laboratory). The E. coli LPS (E. coli LPS 0111:B4, TLR4 agonist, Invivogen) and synthetic lipoprotein tripalmitoyl-S-glyceryl-cysteine (Pam3CSK4; TLR1/2 agonist, Invivogen) were used as controls. The Pam3CSK4 was used after an overnight incubation at 37 °C with lipase from a Burkholderia sp. (Sigma-Aldrich, St. Louis, MO) to exclude lipid.

Kajiwara K., Takata S., To T.T., Takara K., Hatakeyama Y., Tamaoki S., Darveau R.P., Ishikawa H, & Sawa Y. (2017). The promotion of nephropathy by Porphyromonas gingivalis lipopolysaccharide via toll-like receptors. Diabetology & Metabolic Syndrome, 9, 73.

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Biochemical Characterization of Cell Lines

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HEK293 cells (ATCC number CRL-1573) and RAW264.7 cells (ATCC number TIB-71) were acquired from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Dimethyl sulfoxide (DMSO), carboxymethylcellulose (CMC), 3-(4,5-dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyethylenimine (PEI), sodium dodecyl sulfate (SDS), ranitidine, dexamethasone (dexa), L-NAME, kaempferol, genistin, apigenin, lipopolysaccharide (LPS, E. coli 0111:B4), pam3csk4, and poly (I:C) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). RPMI 1640, DMEM, trypsin, PBS, and penicillin-streptomycin were received from HyClone (Logan, UT, USA). Fetal bovine serum (FBS) was acquired from Biotechnics Research, Inc. (Irvine, CA, USA). TRIzol reagent was purchased from MRCgene (Cincinnati, OH, USA). Primers used for semiquantitative RT-PCR and qPCR were purchased from Macrogen Inc. (Seoul, Korea). Phospho-specific or total-protein antibodies against p65, p50, IκBα, PI3K/p85, Src, c-Fos, c-Jun, JNK, ERK, p38, MKK4, MKK7, MEK1/2, TAK1, IRAK1, IRAK4, hemagglutinin (HA), and β-actin were acquired from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). Constructs for signaling proteins HA-Src and HA-TAK1 were used as previously reported [24 (link),26 (link)].

Rahmawati L., Aziz N., Oh J., Hong Y.H., Woo B.Y., Hong Y.D., Manilack P., Souladeth P., Jung J.H., Lee W.S., Jeon M.J., Kim T., Hossain M.A., Yum J., Kim J.H, & Cho J.Y. (2021). Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages, HCl/EtOH-induced Gastritis, and LPS-induced Lung Injury via Attenuation of Src and TAK1. Molecules, 26(19), 6073.

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Isolation and Characterization of Inflammasome Activation

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Kynurenic acid (K3375, purity≥98%) was obtained from Sigma−Aldrich company (Missouri, USA). M-CSF was obtained from NOVUS (Colorado, USA). LPS, nigericin, ATP, MSU, Pam3CSK4, PMA, and poly(A/T) were obtained from Sigma−Aldrich (Missouri, USA). MitoTracker Red, MitoSOX and Lipofectamine 2000 were obtained from Invitrogen (California, USA). Flou-4 AM and DAPI were obtained from Beyotime (Shanghai, China). The Salmonella typhimurium strain ATCC-14028 was obtained from Beina Chuanglian Biotech Co., Ltd. (Beijing, China).

Sun T., Xie R., He H., Xie Q., Zhao X., Kang G., Cheng C., Yin W., Cong J., Li J, & Wang X. (2022). Kynurenic acid ameliorates NLRP3 inflammasome activation by blocking calcium mobilization via GPR35. Frontiers in Immunology, 13, 1019365.

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Murine Dendritic Cell Isolation and Activation

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Pam3CSK4, phorbol 12-myristate 13-acetate (PMA) and ionomycin were purchased from Sigma Aldrich (St. Louis, MO, USA). Dispase, collagenase, DNase, CellROX^TMGreen, FluoReporter™ fluorescein isothiocyanate (FITC), a Protein Labeling Kit, Ovalbmin (OVA), and FITC-conjugated OVA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Percoll was purchased from GE Healthcare (Chicago, IL, USA). A Cytofix/Cytoperm kit with GolgiStop^TM was purchased from BD Bioscience (Franklin Lakes, NJ, USA). Recombinant murine granulocyte macrophage-colony stimulating factor (rmGM-CSF) and recombinant murine interleukin-2 (rmIL-2) were purchased from Peprotech (Rocky Hill, NJ, USA). Anti-CD11c (N418), anti-CD11b (M1/70), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-MHC-II (I-A/I-E; M5/114.15.2), anti-mouse Ly-6G/Ly-6C (Gr-1) (RB6-8C5), anti-CD45 (30-F11), anti-CD3 (17A2), anti-CD4 (GK1.5), anti-Interferon gamma (IFN-γ) (XMG1.2), anti-IL-17A (BL168), and anti-CD16/CD32 (2.4G2) (93) were purchased from Biolegend (San Diego, CA, USA). Anti-CD4 (GK1.5), anti-IFN-γ (H22), anti-IL4 (BVD6-24G2), anti-IL-17A (17F3) mAb, and anti-Ly-6G (1A8) were purchased from Bio X Cell (West Lebanon, NH, USA). The isotype-matched control for each antibody was purchased from the same company.

Peng Z., Cao D.Y., Wu H.Y, & Saito S. (2020). Immunization with a Bacterial Lipoprotein Establishes an Immuno-Protective Response with Upregulation of Effector CD4+ T Cells and Neutrophils Against Methicillin-Resistant Staphylococcus aureus Infection. Pathogens, 9(2), 138.

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