To monitor human TLR3 activation by PSCA-specific polyplexes loaded with Survivin siRNA, the HEK-Blue detection system was used as described previously [25 (link)]. Briefly, 5 × 104 HEK-BluehTLR3/PSCA cells were incubated with PSCA-specific polyplexes based on 25 pmol siRNA. The TLR3 ligand poly(I:C) (1 µg/mL, Sigma-Aldrich) was included as positive control. After 24 h, hydrolysis of secreted alkaline phosphatase (SEAP) color substrate (InvivoGen) by SEAP was quantified at 655 nm using Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments).
Poly 1 c tlr3 ligand
Manufactured by Merck Group
Sourced in United States
Poly(I:C) is a synthetic double-stranded RNA (dsRNA) compound that acts as a Toll-like receptor 3 (TLR3) ligand. It is a laboratory tool used to stimulate the innate immune response by activating the TLR3 signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant mouse ifn γ, by R&D Systems (4 mentions)
Synergy 2 multi mode microplate reader, by Agilent Technologies (1 mentions)
P24 elisa kit, by ZeptoMetrix (1 mentions)
Maxima sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Trisure, by Meridian Bioscience (1 mentions)
Ariamx instrumentation, by Agilent Technologies (1 mentions)
Zymosan, by Thermo Fisher Scientific (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Ovcar3, by American Type Culture Collection (1 mentions)
Caov3, by American Type Culture Collection (1 mentions)
Dmem medium, by Corning (1 mentions)
Pictilisib, by Selleck Chemicals (1 mentions)
Skov3, by Corning (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
Caov3, by Corning (1 mentions)
Ovcar 3, by Corning (1 mentions)
Lps tlr4 ligand, by Merck Group (1 mentions)
9 protocols using poly 1 c tlr3 ligand
1
Monitoring TLR3 Activation by PSCA-Polyplexes
2
Curcumin Modulates HIV-1 Infection and TLR Response
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5x105 chronically HIV-infected H9 T-cells were exposed once or daily to 5 or 50 μM curcumin or serum-free RPMI as control At several time points post-treatment, supernatants were collected and HIV-1 p24-antigen was measured using a commercial p24 ELISA kit (Zeptometrix Corp., Buffalo, NY, USA), as per the manufacturer’s instructions. Alternatively, 1x105 chronically infected H9 T-cells were treated with 5 or 50 μM curcumin for 1 hour and subsequently exposed to TLR ligands for 24 hours. TLR ligands included the TLR3 ligand poly I:C (25 μg/mL; Sigma-Aldrich), the TLR4 ligand LPS from Escherichia coli (100 μg/mL Sigma-Aldrich) and the TLR5 ligand flagellin from Salmonella typhimurium (10 μg/mL; Alpha Diagnostic, San Antonio, TX, USA). TLR ligand concentrations were selected according to previous studies, described elsewhere [5 (link), 19 (link), 33 (link)].
3
Innate Immune Response to Virus Infection
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Non- and PUUV-Suo infected Mygla.REC.B cells were exogenously stimulated with TLR-3 ligand polyI:C (10 µg/mL, Sigma-Aldrich) or Sendai virus (multiplicity of infection 1; received from Prof. Ilkka Julkunen; National Institute of Health and Welfare, Helsinki, Finland). Cells were collected 1-day post-treatment, and RNA was extracted from cell cultures with Trisure (Bioline, London, UK) following the manufacturers’ instructions. Extracted RNA was then reverse transcribed to complementary DNA (cDNA) using random hexamers and RevertAid reverse transciptase (Thermo Scientific). Relative quantitative PCR was performed with Maxima SYBR Green master mix (Thermo Scientific) using AriaMx instrumentation (Agilent, Palo Alto, CA, USA). Published primer sequences were used to measure the mRNAs levels of the interferon-inducible gene were Mx2, a marker of innate immune activation towards virus infection [22 (link)], and actin, which is commonly used for normalization of mRNA levels between samples [23 (link)]. Fold changes of individual mRNA expression levels relative to mock-infected controls were performed by the comparative CT method [24 (link)].
4
TLR Agonist-Induced Cytokine Responses
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To determine TLR responses, two stimulating dosages of TLR1-9 agonists or media only at a log range (except for TLR4 agonist) were chosen to test the dose-dependent effect on PBMCs (3 × 105) in 100 μl complete media at 37 °C for 24 h: 1.0, 10 ug/ml Pam3CSK4 (TLR1/2 ligand, Invivogen, San Diego, CA); 1.0, 10 ug/ml poly (I:C) (TLR3 ligand, Sigma-Aldrich Co., St. Louis, MO); 1.0 ug/ml LPS (TLR4 ligand, Invitrogen, San Diego, CA); 0.1, 1.0 ug/ml flagellin (TLR5 ligand, Calbiochem Corp., San Diego, CA); 0.1, 1.0 ug/ml zymosan (TLR2/6 ligand, Invitrogen, San Diego, CA); 1.0, 10 ug/ml R848 (TLR7/8 ligand, Invitrogen, San Diego, CA); and 1.0, 10 ug/ml ODN 2216 (TLR9 ligand, Coley Pharmaceuticals Wellesley, MA). IL-6, IL-8, IL-10, IL-12, TNF-α., IFN-γ (R & D Systems, Minneapolis, MN), INF-α and INF-β (TFB, Fujirebio, Inc., Tokyo) production was assessed by ELISA as previously described [26 (link),27 (link)]. Every condition was performed in duplicate. P < 0.05 was thought of significance by unpaired t tests (Graph Pad 4.0).
5
BM-derived MSC Isolation and Priming
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The BM-derived MSCs were isolated from C57BL/6 mice and expanded, as described in our previous work [19 (link)]. In brief, BM cells were flushed out from femurs and tibias, plated in 75 cm2 tissue culture flasks at a concentration of 1 × 106 cells/mL in the complete culture medium, and incubated at 37 °C and 5% CO2. Non-adherent cells were removed after 3 days, and the remaining cells were passaged into a new flask when the cells reached 70~80% confluency. To do priming, we harvested cells at the 90% confluency and plated them in 12-well plates (5 × 104 cells/well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA). Poly(I:C) (TLR3 ligand, 10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
6
Priming of BM-derived MSCs with IFN-γ and Poly(I:C)
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The BM-derived MSCs were isolated from C57BL/6 mice and expanded, as described in our previous work (19) .
In brief, BM cells were flushed out from femurs and tibias, plated in 75 cm 2 tissue culture flasks at a concentration of 1×10 6 cells/mL in the complete culture medium, and incubated at 37 and 5% CO 2 . Nonadherent cells were removed after three days, and the remaining cells were passaged into a new flask when the cells reached 70~80% confluency. To do priming, we harvested cells at the 90% confluency and plated them in 12-well plates (5×10 4 cells/ well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA). Poly(I:C) (TLR3 ligand, 10 μg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
In brief, BM cells were flushed out from femurs and tibias, plated in 75 cm 2 tissue culture flasks at a concentration of 1×10 6 cells/mL in the complete culture medium, and incubated at 37 and 5% CO 2 . Nonadherent cells were removed after three days, and the remaining cells were passaged into a new flask when the cells reached 70~80% confluency. To do priming, we harvested cells at the 90% confluency and plated them in 12-well plates (5×10 4 cells/ well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA). Poly(I:C) (TLR3 ligand, 10 μg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
7
Priming of BM-derived MSCs for Immune Modulation
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The BM-derived MSCs were isolated from C57BL/6 mice and expanded, as described in our previous work (19) . In brief, BM cells were ushed out from femurs and tibias, plated in 75 cm 2 tissue culture asks at a concentration of 1×10 6 cells/mL in the complete culture medium, and incubated at 37℃ and 5% CO 2 .
Non-adherent cells were removed after three days, and the remaining cells were passaged into a new ask when the cells reached 70~80% con uency. To do priming, we harvested cells at the 90% con uency and plated them in 12-well plates (5×10 4 cells/ well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA). Poly(I:C) (TLR3 ligand, 10 μg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
Non-adherent cells were removed after three days, and the remaining cells were passaged into a new ask when the cells reached 70~80% con uency. To do priming, we harvested cells at the 90% con uency and plated them in 12-well plates (5×10 4 cells/ well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA). Poly(I:C) (TLR3 ligand, 10 μg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
8
Priming BM-derived MSCs with IFN-γ and Poly(I:C)
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The BM-derived MSCs were isolated from C57BL/6 mice and expanded, as described in our previous work (19) . In brief, BM cells were ushed out from femurs and tibias, plated in 75 cm 2 tissue culture asks at a concentration of 1 × 10 6 cells/mL in the complete culture medium, and incubated at 37℃ and 5% CO 2 .
Non-adherent cells were removed after three days, and the remaining cells were passaged into a new ask when the cells reached 70 ~ 80% con uency. To do priming, we harvested cells at the 90% con uency and plated them in 12-well plates (5 × 10 4 cells/ well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA).
Poly(I:C) (TLR3 ligand, 10 µg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
Non-adherent cells were removed after three days, and the remaining cells were passaged into a new ask when the cells reached 70 ~ 80% con uency. To do priming, we harvested cells at the 90% con uency and plated them in 12-well plates (5 × 10 4 cells/ well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA).
Poly(I:C) (TLR3 ligand, 10 µg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
9
Ovarian Cancer Cell Lines and Reagents
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Cell lines. The human ovarian cancer cell lines Caov-3, OVCAR-3, OV-90, and SK-OV-3 were purchased from the ATCC (Manassas, VA, uSA). Caov-3 and OVCAR-3 cells, representing primary cancer, were harvested from human ovarian adenocarcinoma confined to the ovary. OV-90 and SK-OV-3 cells, as representative metastatic cancer, were established from ascites derived from ovarian cancer patients. Caov-3 and OV-90 cells were maintained in DMEM medium (Corning Incorporated, Corning, NY, uSA) supplemented with 10% FBS (RMBIO, Missoula, MT, uSA), penicillin, streptomycin, and glutamine at 37˚C in 5% CO 2 . OVCAR-3 and SK-OV-3 cells were maintained in RPMI-1640 medium (Corning Inc.) supplemented with 10% FBS (RMBIO), penicillin, streptomycin, and glutamine at 37˚C in 5% CO 2 .
Chemicals. LPS (TLR4 ligand) and poly (I:C) (TLR3 ligand) were obtained from Sigma-Aldrich (St. Louis, MO, uSA). MALP-2 (TLR2/6 ligand) was purchased from Enzo Life Sciences (Farmingdale, NY, uSA). PP1 (Src inhibitor) and Bay 61-3606 (Syk inhibitor) were purchased from Calbiochem (San Diego, CA, USA). A66 (p110α inhibitor), TGX-221 (p110β inhibitor), CAL-101 (p110δ inhibitor), LY294002 (pan-p110 inhibitor), Bay 80-6946 (p110α and p110β inhibitor), and pictilisib (p110α and p110δ inhibitor) were obtained from Selleckchem (Houston, TX, uSA). Recombinant Gal-1 was purchased from R&D Systems (Minneapolis, MN, uSA).
Chemicals. LPS (TLR4 ligand) and poly (I:C) (TLR3 ligand) were obtained from Sigma-Aldrich (St. Louis, MO, uSA). MALP-2 (TLR2/6 ligand) was purchased from Enzo Life Sciences (Farmingdale, NY, uSA). PP1 (Src inhibitor) and Bay 61-3606 (Syk inhibitor) were purchased from Calbiochem (San Diego, CA, USA). A66 (p110α inhibitor), TGX-221 (p110β inhibitor), CAL-101 (p110δ inhibitor), LY294002 (pan-p110 inhibitor), Bay 80-6946 (p110α and p110β inhibitor), and pictilisib (p110α and p110δ inhibitor) were obtained from Selleckchem (Houston, TX, uSA). Recombinant Gal-1 was purchased from R&D Systems (Minneapolis, MN, uSA).
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