Three hundred and eighty microliters of fresh whole blood in citrate mixed 1∶1 with RPMI media was added to pre-prepared plates containing 20 µL of Pam3CSK4 (Invivogen, San Diego, CA) for a final concentration of 100 ng/mL. Pam3CSK4 is a specific agonist for TLR2/1. Plates were incubated at 37°C on a shaking incubator under 5% CO2 for 6 h before being spun down and plasma removed and frozen at −80°C. IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β were later assayed in duplicate on a multiplex bead system (Luminex, Austin, TX) using reagents from R&D Systems (Minneapolis, MN). A complete blood count with differential was performed in the hospital clinical laboratory for each subject at the time of phlebotomy.
Pam3csk4
Manufactured by InvivoGen
Sourced in United States, France, United Kingdom, Canada, Germany, Sweden, Italy, Macao
Pam3CSK4 is a synthetic triacylated lipopeptide that mimics the structure of the acylated amino terminus of bacterial lipoproteins. It acts as a potent agonist of Toll-like receptor 2 (TLR2) and can be used in cell-based assays to study TLR2-mediated cellular responses.
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Lab products found in correlation
Poly 1 c, by InvivoGen (67 mentions)
Lipopolysaccharides (lps), by Merck Group (67 mentions)
Lipopolysaccharides (lps), by InvivoGen (66 mentions)
Pam2csk4, by InvivoGen (51 mentions)
Fsl 1, by InvivoGen (43 mentions)
Flagellin, by InvivoGen (38 mentions)
Imiquimod, by InvivoGen (31 mentions)
Nigericin, by Merck Group (17 mentions)
Adenosine triphosphate (atp), by Merck Group (17 mentions)
Zymosan, by InvivoGen (16 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (15 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (14 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (14 mentions)
Ultrapure lps, by InvivoGen (12 mentions)
Tnf α, by Thermo Fisher Scientific (12 mentions)
Opti mem, by Thermo Fisher Scientific (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Phorbol myristate acetate (pma), by Merck Group (11 mentions)
Poly 1 c hmw, by InvivoGen (11 mentions)
Poly 1 c, by Merck Group (10 mentions)
641 protocols using pam3csk4
1
Whole Blood Cytokine Profiling
2
Modulating Laser-Induced CNV with PAM3CSK4 and 4G3 Antibody
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PAM3CSK4 is a synthetic diacylated lipopeptide activating TLR-2 with cooperation from TLR-1. PAM3CSK4 (InvivoGen, San Diego, CA) was dissolved in sterile endotoxin free water to a final concentration of 0.5 mg/mL. Mice were injected intraperitoneally (i.p.) with 50 μg of PAM3CSK4 (henceforth referred to as PAM) solution or control [sterile endotoxin free water or sterile phosphate buffered saline (PBS)]. In time course studies of laser-induced CNV area, a single dose of PAM was injected at either day 0, 1, 2, 3, or 4 relative to laser application. For subsequent studies, single i.p. injections of PAM or vehicle control (either sterile water or PBS) were administered 2 days after laser application.
4G3 is a human Fab converted to a full-length IgG antibody with a mouse IgG1 Fc tail.38 (link) 4G3 binds to mouse VEGF164 with a dissociation constant of 10 pM and neutralizes mouse VEGF binding to human VEGFR2 with an EC50 of 0.15 nM in a binding assay (ELISA; MSD, Rockville, MD).38 (link) For efficacy studies in the mouse laser CNV assay, 4G3 dissolved in PBS was dosed i.p. at doses of 0.1, 0.3, 1.0, 3.0, or 10 mg/kg at day 0, 2, and 4 after laser. Either rat IgG2a isotype (MAB006; R&D systems, McKinley Place, MN) dissolved in PBS or PBS was dosed i.p. as negative controls for laser CNV studies.
4G3 is a human Fab converted to a full-length IgG antibody with a mouse IgG1 Fc tail.38 (link) 4G3 binds to mouse VEGF164 with a dissociation constant of 10 pM and neutralizes mouse VEGF binding to human VEGFR2 with an EC50 of 0.15 nM in a binding assay (ELISA; MSD, Rockville, MD).38 (link) For efficacy studies in the mouse laser CNV assay, 4G3 dissolved in PBS was dosed i.p. at doses of 0.1, 0.3, 1.0, 3.0, or 10 mg/kg at day 0, 2, and 4 after laser. Either rat IgG2a isotype (MAB006; R&D systems, McKinley Place, MN) dissolved in PBS or PBS was dosed i.p. as negative controls for laser CNV studies.
3
Stimulation of Alveolar Macrophages
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Alveolar macrophages in supplemented RPMI-1640 media were plated out in 96- and 24-well plates at a concentration of 1×106/mL. Cells were incubated with LPS (Sigma-Aldrich, serotype O26:B6; 1 μg/mL) or Pam3CSK4 (Sigma-Aldrich; 0.1 μg/mL) for a 24-hour period before removing supernatants and washing 3 times with media. Cells were then stimulated again with LPS (1 μg/mL) or Pam3CSK4 (0.1 μg/mL) for 24 hours or as indicated; these concentrations have been shown to give optimal stimulation of alveolar macrophages at 24 hours.17 (link),25 (link) For TLR mRNA analysis, cells were stimulated with LPS (1 μg/mL), Pam3CSK4 (0.1 μg/mL), or ultrapure Escherichia coli O111:B4 LPS (UPLPS; InvivoGen, San Diego, CA, USA; 0.1 μg/mL) for 4, 6, 24, and 48 hours. Where necessary, supernatants were removed and frozen for future cytokine analysis.
4
Immobilized ligand pull-down assay for HyCaspA
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Biotin-conjugated lipid A (10 μg), LPS (50 μg), Pam3CSK4 (50 μg), and MDP (50 μg) from E. coli O111:B4 (InvivoGen) were immobilized onto 8 μl of streptavidin Sepharose beads (Thermo Fisher Scientific). For pull-down assay, the beads were washed three times in lysis buffer to remove unconjugated ligands and incubated with transfected cell lysates at 4°C for 12 hours. For the competition assay, 20 μg of unlabeled LPS, lipid A, Pam3CSK4, or MDP from E. coli O111:B4 (InvivoGen) was incubated with cell lysates for 60 min at room temperature before adding biotinylated LPS-conjugated streptavidin beads. The beads were washed in lysis buffer, and the precipitates were eluted in 1 × SDS loading buffer followed by immunoblotting analysis. To evaluate LPS binding to endogenous HyCaspA, 120 to 150 polyps were pretreated with 100 μM Z-VAD-FMK (Sigma-Aldrich) for 2 hours and lysed in the Hydra lysis buffer. For standard pull-down assay, 50 μg of biotin-conjugated LPS, lipid A, or MDP was immobilized onto streptavidin Sepharose beads followed by incubation with Hydra lysates. To examine LPS binding to purified recombinant HyCasps, 1 μg of Flag-purified HyCasp proteins (catalytic-cysteine mutants) was added to 250 μl of cell lysis buffer to perform pull-down assay.
5
Murine Intestinal Epithelial Cell Stimulation
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The murine intestinal epithelial cell line CT26 (ATCC; CRL-2638) was used. CT26 cells were cultured with complete RPMI medium containing 10% heat-inactivated foetal bovine serum, 100 mg/ml streptomycin, and 100 mg/ml penicillin. Cells were incubated in a 5% CO2 incubator at 37 °C. For stimulation trials, CT26 cells were seeded at 1 × 106 cells/well in 6-well culture plates. The stimulators were added as follows: Pam3CSK4 (TLR2 ligand) 1 μg/ml, LPS (TLR4 ligand) 100 ng/ml, ODN1585 (TLR9 ligand) 100 nM, IFN-γ 10 ng/ml, TNF-α 100 ng/ml, IL-1β 10 ng/ml, and IL-17A 200 ng/ml (Pam3CSK4 and ODN1585 from InvivoGen [San Diego, CA, USA], LPS from Sigma-Aldrich [Saint Louis, MO, USA], and others from R&D Systems). Cells were stimulated for 24 hours before supernatants were harvested, and stored at −80 °C.
6
Isolation and Activation of T Cell Subsets
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Splenocytes and lymphnodes were harvested from 8- to 12-week-old mice and pooled. CD4 cells were MACS (magnetic assisted cell sorting) purified by using CD4 isolation kit II (Miltenyi Biotec, Auburn, CA) or sorted using EasySep kits. Purified CD4 cells were FACS (Fluorescence Activated Cell Sorting) sorted for CD4+CD25−GFP− Tcon cells or CD4+CD25+ GFP+ Treg cells (>99% purity). In some experiments CD25+ Tregs and CD4+CD25− CD44l°w naïve Tcon cells were sorted using EasySep kits, and the purity of Tregs was more than 92%. 6 × 104 FACS sorted Tcons or Tregs were cultured in U-bottom 96 well plates with 2 ng/mL IL-2 only, or also with TLR ligands. Pam3CSK4, heat killed Candida albicans (HKCA), Lipopolysaccharide (LPS), and Flagellin were purchased at Invivogen and were used at following concentrations: LPS, 1 ug/mL; Flagellin, 1μg/mL; Pam3CSK4, 5 μg/mL and HKCA, 106/mL. In some experiments, formaldehyde fixed Candida albicans germtube (FCA) were used at 106/mL.
7
Epithelial Cell Priming and Infection
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Where indicated, cells were primed with 100ng/ml or 400ng/ml of Pam3CSK4 (Invivogen) for 3 hours prior to infection. To induce SPI-1 expression, overnight cultures of Salmonella were diluted into LB broth containing 300 mM NaCl and grown for 3 hours standing at 37°C (28) . Overnight cultures of Listeria were diluted and grown shaking for Cell culture of intestinal epithelial cell lines All cell lines were obtained from American Type Culture Collection (ATCC).
Caco-2 cells (HTB-37; ATCC) were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. T84 cells (CCL-248; ATCC) were maintained in DMEM F-12 supplemented with 5% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
One day prior to infection or treatment, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) diluted 1:1 with 1X PBS. Cells were incubated with trypsin at 37°C for 15 minutes, after which the trypsin was neutralized with serum-containing media. Cells were replated in media without antibiotics in a 24-well plate at a concentration of 3 × 10 5 cells/well. Where indicated, cells were primed with 100ng/mL or 400ng/ml Pam3CSK4 (Invivogen) or 500ng/ml LPS (Sigma-Aldrich) for 3h or 16h prior to anthrax toxin treatment or bacterial infections.
Caco-2 cells (HTB-37; ATCC) were maintained in DMEM supplemented with 10% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. T84 cells (CCL-248; ATCC) were maintained in DMEM F-12 supplemented with 5% (vol/vol) heat-inactivated FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin.
One day prior to infection or treatment, cells were dissociated with 0.25% Trypsin-EDTA (Gibco) diluted 1:1 with 1X PBS. Cells were incubated with trypsin at 37°C for 15 minutes, after which the trypsin was neutralized with serum-containing media. Cells were replated in media without antibiotics in a 24-well plate at a concentration of 3 × 10 5 cells/well. Where indicated, cells were primed with 100ng/mL or 400ng/ml Pam3CSK4 (Invivogen) or 500ng/ml LPS (Sigma-Aldrich) for 3h or 16h prior to anthrax toxin treatment or bacterial infections.
8
Cytokine Stimulation of PBMCs
9
Neuroprotective effects of Pam3CSK4 and siTLR2 in OGD-injured PC12 cells
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For Pam3CSK4 treatment, normal PC12 cells were treated with 300 ng/ml Pam3CSK4 (InvivoGen at the time of OGD injury, and cells treated with an equal volume of PBS served as the controls. For the Ad-siTLR2 group, normal PC12 cells at 80 % confluence were infected with siTLR2 adenovirus [40 (link)] for 24-48 h, and PC12 cells subjected to an equivalent infection load of RFP-expressing adenovirus served as the controls.
For the three OGD groups that were subjected to different treatments, the PC12 cells were subjected to OGD for 6 h after Pam3CSK4 treatment or after siTLR2 infection for 24 h. Untreated PC12 cells that were subjected to OGD were used as negative controls. The PC12 cells that were treated with Pam3CSK4 or siTLR2 were co-cultured with MSCs for an additional 24 h following OGD. Untreated cells were exposed to OGD and co-cultured with MSCs for use as negative controls for the other MSC co-culture groups.
For the three OGD groups that were subjected to different treatments, the PC12 cells were subjected to OGD for 6 h after Pam3CSK4 treatment or after siTLR2 infection for 24 h. Untreated PC12 cells that were subjected to OGD were used as negative controls. The PC12 cells that were treated with Pam3CSK4 or siTLR2 were co-cultured with MSCs for an additional 24 h following OGD. Untreated cells were exposed to OGD and co-cultured with MSCs for use as negative controls for the other MSC co-culture groups.
10
Cytokine Production in Human and Mouse DCs
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Human iPS-DCs were plated at 2 × 104 cells per well in 200 μl of X-VIVO-15 medium without cytokines. TLR ligands were added directly to the medium, and supernatants were harvested after a 24 h incubation at 37 °C. For the assays, TLR ligands were used at the following concentrations: TLR2; Pam3CSK4, 300 ng/ml (InvivoGen); TLR3; Poly(I:C), 50 μg/ml (InvivoGen); TLR4; Lipopolysaccharide (LPS), 500 ng/ml (Sigma-Aldrich); TLR7; Imiquimod, 50 μg/ml (InvivoGen); and TLR9; ODN 2216, 3 μg/ml (Miltenyi Biotech). Mouse BM-DCs were plated out at 1 × 105 cells per well. TLR ligands were added directly to the well as before with supernatants harvested at 6 and 24 h at 37 °C. TLR ligands were used at the following concentrations: TLR2; Pam3CSK4, 0.5 μg/ml (InvivoGen); TLR3; Poly(I:C), 10 μg/ml (InvivoGen); TLR4; LPS, 10 μg/ml (InvivoGen); TLR5; Flagellin 5 μg/ml (InvivoGen); TLR7; Imiquimod, 5 μg/ml (InvivoGen); and TLR9; CpG Class B ODN 1826, 0.05 μM (Invivogen).
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