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Pam3cks4

Manufactured by InvivoGen
Sourced in United States

Pam3CKS4 is a synthetic triacylated lipopeptide that mimics the structure of bacterial lipoproteins. It acts as a Toll-like receptor 2 (TLR2) agonist, triggering the activation of NF-κB and the production of proinflammatory cytokines.

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2 protocols using pam3cks4

1

Whole Blood Cytokine Signaling Assay

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Fresh whole blood (lithium heparin) was incubated with 10 µg/mL polymyxine B (Sigma-Aldrich), CD45-V500 (BD biosciences), and CD14-FITC (BD biosciences) in the absence or presence of 75 ng/mL IL-1b (eBioscience), 1 µg/ml Pam3CKS4 (In vivogen), 3 µg/mL R848 (In vivogen), or 20 ng/mL TNF-a (Peprotech). Cells were incubated for 15 min at 37°C, 5% CO2. Thereafter, the RBCs were lysed, fixed [with Lyse/Fix Buffer (BD biosciences) for 10 minutes at 37°C, 5% CO2], permeabilized (Perm Buffer II, BD biosciences) for 30 minutes on ice, washed with Stain buffer (BD biosciences) and incubated with 1:20 diluted PE Mouse anti-IkBa (BD biosciences).
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2

Heterologous expression of Streptococcal pilus

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Bacterial strains are listed in Table 1. L. lactis MG1363 was grown under static conditions at 28°C, in M17 broth supplemented with 5% glucose and 200 µg mL–1 kanamycin where appropriate. The plasmid containing the complete pilus operon from the genome of S. pyogenes serotype M1/T1 strain SF370 (ATCC 700294) under the control of the lactococcal promoter P2323 was introduced into L. lactis by electroporation as described previously.23Escherichia coli BL21 was grown at 37°C in LB containing 50 µg mL–1 ampicillin and 30 µg mL–1 chloramphenicol. The TLR2 agonist Pam3CKS4, the TLR2/6 agonist Pam2CKS4 and the TLR4 agonist LPS from E. coli K12 strain (LPS‐EK) were obtained from InvivoGen (San Diego, CA, USA). ToxOut Endotoxin‐Free Water from BioVision (San Francisco, CA, USA) was utilized as a negative control and diluent in the cell‐based assays.
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