HEK-BlueTM hTLR cells were seeded in HEK-BlueTM Detection medium into 96-well plates (50,000 cells/well) which were pre-filled with 20 µl culture supernatants of irradiated breast cancer cell lines. LPS (10 ng/ml) or fibroblast-stimulating lipopeptide-1 (FSL-1, Pam2CGDPKHPKSF, 10 ng/ml, InvivoGen) served as positive controls for hTLR4 or hTLR2 activation, respectively. TLR stimulation was allowed for 7 h at 37°C. Subsequently, activity of secreted alkaline phosphatase (SEAP) was measured kinetically in a Synergy MX plate reader (10 h at 37°C, absorption at 630nm, measurement every 2 min at 37°C) and calculated from the slope of the corresponding regression lines. Relative TLR activation was expressed as x-fold values of samples treated with 0 Gy culture supernatants.
Pam2cgdpkhpksf
Manufactured by InvivoGen
Sourced in France
Pam2CGDPKHPKSF is a synthetic lipoprotein that contains a diacylated cysteine (Pam2C) moiety linked to a glycine-lysine dipeptide (GK) and a hexapeptide sequence (HPKSF). This product is commonly used as a Toll-like receptor 2 (TLR2) agonist in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy micro kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Prostaglandin e2 parameter assay kit, by R&D Systems (1 mentions)
Polyinosinic polycytidylic acid poly 1 c, by Merck Group (1 mentions)
Fsl 1, by InvivoGen (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Mtt assay, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Bay11 7085, by Santa Cruz Biotechnology (1 mentions)
Cyt387, by Selleck Chemicals (1 mentions)
Pam3csk4 p3c, by InvivoGen (1 mentions)
U0126, by InvivoGen (1 mentions)
Sp600125, by Selleck Chemicals (1 mentions)
Sb203580, by Selleck Chemicals (1 mentions)
3 protocols using pam2cgdpkhpksf
1
Quantifying TLR Activation by Irradiated Breast Cancer Cells
2
TLR Agonist Stimulation and Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The agonists for TLR2 (heat-killed Listeria monocytogenes, HKLM, Cat tlr1-hklm), and TLR2/6 (FSL-1, Pam2CGDPKHPKSF, Cat tlr1-fsl) were purchased from Invivogen, (Toulouse, France), the TLR3 agonist polyinosinic-polycytidylic acid, Poly I:C, Cat P9582) was purchased from Sigma-Aldrich (Gillingham, UK). Total RNA was extracted using TRIzol™ (Invitrogen Life Technologies) or the RNeasy Micro Kit (Qiagen). All other reagents for cDNA synthesis and quantitative PCR were purchased from Sigma-Aldrich (Gillingham, UK). All primers were from Thermo-Scientific (Waltham, MA). Table 1 shows sequences used for amplification of target genes. Antibodies to detect the phosphorylated p-65 NF-κB subunit Cat #3031 (AB_330559), phosphorylated c-Jun subunit Cat #9164 (AB_330892), phosphorylated IRF3 Cat #29047 (AB_2773013), and ß-actin Cat #A5441 (AB_476744) were purchased from Cell Signalling Technology (Beverly, MA) and Sigma-Aldrich (Gillingham, UK), respectively. The Meso Scale Discovery Immunoassay was used to detect a panel of 9 cytokines and chemokines MSD (Rockville, MD). The Prostaglandin E2 parameter assay kit (KGE004B) was purchased from R&D Systems (Minneapolis, MN).
3
Toll-like Receptor Signaling in GC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!