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Poly 1 c

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Poly I:C is a synthetic analog of double-stranded RNA (dsRNA) that mimics viral infection and induces an innate immune response. It is commonly used in cell biology and immunology research to study antiviral and inflammatory pathways.

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Lab products found in correlation

Lipopolysaccharides (lps), by Merck Group (4 mentions) T1420, by Tokyo Chemical Industry (2 mentions) Cpg odn 1668, by Enzo Life Sciences (2 mentions) H 2kb siinfekl tetramer staining, by Beckman Coulter (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Ova af488, by Thermo Fisher Scientific (1 mentions) Papain, by Merck Group (1 mentions) Gm csf, by CellGenix (1 mentions) Il 1β, by CellGenix (1 mentions) Interleukin 6 (il 6), by CellGenix (1 mentions) Tnf α, by CellGenix (1 mentions) Interleukin 4 (il 4), by CellGenix (1 mentions) Lymphoprep, by Axis-Shield (1 mentions) X vivo medium, by Lonza (1 mentions) Metformin, by Selleck Chemicals (1 mentions) Mdsc isolation kit, by Miltenyi Biotec (1 mentions) Sybr green dye, by Thermo Fisher Scientific (1 mentions) Rapamycin, by Selleck Chemicals (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Recombinant mouse il 6, by Miltenyi Biotec (1 mentions)

13 protocols using poly 1 c

1

Intranasal Delivery of Soluble OVA and Apoptotic Cells

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Intranasal deliveries were performed using an optimized delivery system27 (link),55 (link). Mice were completely anesthetized with avertin using 300 μl per mouse with tert-amyl alcohol content at 2.5% and 2,2,2 tribromoethanol (TCI America, T1420) at a concentration of 50 mg per kg. Soluble OVA, apoptotic cells and TLR agonists were delivered in a 50 μl volume. Final concentration of deliveries were 1 μg soluble OVA (sOVA) (0.22 micron filtered, Grade VII Sigma) or 20 × 106 apoptotic cells ± 50μg R848 (Enzo Life Sciences) or ± 10 μg Poly I:C (Enzo Life Sciences). Mice were sacrificed at various times depending on the experimental design.
Desch A.N., Gibbings S.L., Clambey E.T., Janssen W.J., Slansky J.E., Kedl R.M., Henson P.M, & Jakubzick C. (2014). Dendritic cell subsets require cis-activation for cytotoxic CD8 T cell induction. Nature communications, 5, 4674.
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2

Intranasal Immunization with OVA-AF488 and TLR Adjuvants

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Mice were immunized i.n. with 40 μl of 3 μg OVA-AF488 (#034781; Invitrogen) combined with individual treatments with following different TLR adjuvants: 2 μg LPS (#L8274; Sigma-Aldrich), 50 μg Poly I:C (#ALX-746-021; Enzo), 50 μg R848 (#ALX-420-038; Enzo), 20 μg CpG/ODN1668 (#ALX-746-051; Enzo), and non-TLR adjuvant: 8 μg Papain (#76216; Sigma-Aldrich). i.n. immunization with CFSE alone was performed at a concentration of 5 μM in 40 μl. WT mice were immunized with 3 μg OVA-AF488 combined with TLR adjuvant ± pertussis toxin (2 μg/mouse). After 24 h, LLNs were harvested and analyzed for Ag+-positive myeloid cells.
Rawat K., Tewari A., Li X., Mara A.B., King W.T., Gibbings S.L., Nnam C.F., Kolling F.W., Lambrecht B.N, & Jakubzick C.V. (2023). CCL5-producing migratory dendritic cells guide CCR5+ monocytes into the draining lymph nodes. The Journal of Experimental Medicine, 220(6), e20222129.
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3

Assessing OT-I T Cell Responses

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First, we performed a dose dependent analysis of OT-I T cell proliferation response to Poly I:C and R848. 10 μg for Poly I:C and 50μg for R848 were selected based on their capacity to induce quantitatively similar OT-I T cell proliferation (Supplementary Fig. 10). One day after OT-I cell adoptive transfer, WT mice were administered by intranasal delivery with 1 μg soluble OVA (0.22 micron filtered, Grade VII Sigma) WT-apoptotic or OVA-apoptotic cells ± 10 μg Poly I:C (Enzo Life Sciences) or 50 μg R848 (Enzo Life Sciences). Five days after immunization, in vivo target cells were labeled using 15 μM PBSE. 107 PBSE-labeled CD45.1 WT and CD45.2 OVA-expressing splenocytes (derived from ACTB-OVA mice) were injected at a (1:1) ratio i.v. The following day, spleens were harvested as described and specific killing of adoptively transferred congenic mismatched populations was assessed by flow cytometry.
Desch A.N., Gibbings S.L., Clambey E.T., Janssen W.J., Slansky J.E., Kedl R.M., Henson P.M, & Jakubzick C. (2014). Dendritic cell subsets require cis-activation for cytotoxic CD8 T cell induction. Nature communications, 5, 4674.
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4

Dendritic Cell and Islet Cell Interaction

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mDCs were left unstimulated, or stimulated with poly I:C (Enzo Life Sciences)(20μg/ml), or were co-cultured in a 1:1 ratio with mock- or CVB3-infected Min6 cells. Alternatively, mDCs were exposed to CVB3 (MOI of 50) in X-Vivo15. Supernatant and cells were harvested at indicated times for mRNA isolation, flow cytometry or ELISA/bead array, or used in allogeneic mixed lymphocyte reaction (MLR).
Schulte B.M., Gielen P.R., Kers-Rebel E.D., Schreibelt G., van Kuppeveld F.J, & Adema G.J. (2015). Enterovirus-Infected β-Cells Induce Distinct Response Patterns in BDCA1+ and BDCA3+ Human Dendritic Cells. PLoS ONE, 10(3), e0121670.
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5

Generating Mature Dendritic Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood (Sanquin, Nijmegen, Netherlands) by density gradient centrifugation using lymphoprep (Axis-Shield). Adherent cells were obtained as previously described [32 (link)]. Immature DCs were generated by culturing the adherent cells in X-VIVO medium (Lonza) containing 2% human serum (Sanquin), with IL-4 (300 U/ml) and GM-CSF (450 U/ml; both CellGenix) for 6 days. On day 6, DCs were maturated with either a cytokine cocktail containing prostaglandin E2 (PGE2; 10 μg/ml; Pfizer), tumor necrosis factor (TNF)-α (10 ng/ml), IL-1β (5 ng/ml), and IL-6 (15 ng/ml; all CellGenix) or with toll-like receptor (TLR)3 and TLR7/8 ligands, poly[I:C] (20 μg/ml; Enzo), and R848 (4 μg/ml; InvivoGen), respectively, for 24 hours.
de Haas N., de Koning C., di Blasio S., Flórez-Grau G., de Vries I.J, & Hato S.V. (2019). STAT Family Protein Expression and Phosphorylation State during moDC Development Is Altered by Platinum-Based Chemotherapeutics. Journal of Immunology Research, 2019, 7458238.
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6

Assessing OT-I T Cell Responses

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First, we performed a dose dependent analysis of OT-I T cell proliferation response to Poly I:C and R848. 10 μg for Poly I:C and 50μg for R848 were selected based on their capacity to induce quantitatively similar OT-I T cell proliferation (Supplementary Fig. 10). One day after OT-I cell adoptive transfer, WT mice were administered by intranasal delivery with 1 μg soluble OVA (0.22 micron filtered, Grade VII Sigma) WT-apoptotic or OVA-apoptotic cells ± 10 μg Poly I:C (Enzo Life Sciences) or 50 μg R848 (Enzo Life Sciences). Five days after immunization, in vivo target cells were labeled using 15 μM PBSE. 107 PBSE-labeled CD45.1 WT and CD45.2 OVA-expressing splenocytes (derived from ACTB-OVA mice) were injected at a (1:1) ratio i.v. The following day, spleens were harvested as described and specific killing of adoptively transferred congenic mismatched populations was assessed by flow cytometry.
Desch A.N., Gibbings S.L., Clambey E.T., Janssen W.J., Slansky J.E., Kedl R.M., Henson P.M, & Jakubzick C. (2014). Dendritic cell subsets require cis-activation for cytotoxic CD8 T cell induction. Nature communications, 5, 4674.
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7

Synergistic Immunomodulatory Therapy Assessment

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INK128, rapamycin, and metformin were purchased from Selleckchem Inc. Recombinant mouse IFN-α2, anti-CD11b-fluorescein isothiocyanate (FITC), anti-Gr1-allophycocyanin (APC), anti-Ly6G-phycoerythrin (PE), and anti-Ly6C-APC were purchased from Biolegend Inc. TRIzol reagent and SYBR green dye were purchased from Invitrogen Inc. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Inc. Collagenase type D and DNase I were purchased from Roche Inc. Pristane, N-methyl-2-pyrrolidone (NMP), and polyvinyl pyrrolidone (PVP) were purchased from Sigma Inc. Antibodies for α-tubulin (2144), p-S6 (4858S), S6 (2217S), p-4EBP-1 (2855S), 4EBP-1 (9644T), p-mTOR (5536T), p-AMPK (25375), and IRF-8 (83413T) were purchased from Signal Technology Inc. MDSC isolation kit, recombinant mouse IL-6, and GM-CSF were purchased from Miltenyi Biotec Inc. R848, CpG, poly I:C, and TNF-ɑ were purchased from Enzo Life Science Inc. Mouse albumin enzyme linked immunosorbent assay (ELISA) quantitation set, mouse anti-IgG, and anti-dsDNA IgG kit were purchased from Bethyl Laboratories Inc.
Shi G., Li D., Zhang D., Xu Y., Pan Y., Lu L., Li J., Xia X., Dou H, & Hou Y. (2021). IRF-8/miR-451a regulates M-MDSC differentiation via the AMPK/mTOR signal pathway during lupus development. Cell Death Discovery, 7, 179.
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8

Isolation and Stimulation of Thioglycollate-Elicited Macrophages

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Four days after intraperitoneal injection of 2 mL of BBL thioglycollate medium [4% (wt/vol) brewer’s thioglycollate medium power (BD Biosciences)], thioglycollate-elicited macrophages were recovered in 5 mL of PBS by peritoneal lavage. Cells were seeded onto 96-well plates at 1 × 105 cells per well and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco), 1% penicillin/streptomycin (Life Technologies) at 37 °C, and 5% CO2. Macrophages were stimulated for 4 h with Pam3CSK4 (40 ng/mL), poly(I:C) (20 μg/mL), lipopolysaccharide (LPS) (10 ng/mL), R848 (25 ng/mL) (all from Enzo Life Science), or CpG (Sigma-Aldrich, 100 μg/mL). dsDNA (IDT, 2 μg/mL) was complexed with Lipofectamine 2000 (Life Technologies) and transfected according to the manufacturer’s instructions. Nigericin (Sigma-Aldrich, 10 μg/mL) stimulation for 1 h was performed after 4-h LPS priming (10 ng/mL). Cytokine concentrations in the supernatants were measured using ELISA kits for mouse IFN-α, interleukin-1β, and TNF (eBioscience).
Zhong X., Choi J.H., Hildebrand S., Ludwig S., Wang J., Nair-Gill E., Liao T.C., Moresco J.J., Liu A., Quan J., Sun Q., Zhang D., Zhan X., Choi M., Li X., Wang J., Gallagher T., Moresco E.M, & Beutler B. (2022). RNPS1 inhibits excessive tumor necrosis factor/tumor necrosis factor receptor signaling to support hematopoiesis in mice. Proceedings of the National Academy of Sciences of the United States of America, 119(18), e2200128119.
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9

Intranasal Delivery of Soluble OVA and Apoptotic Cells

Cited 1 time
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Intranasal deliveries were performed using an optimized delivery system27 (link),55 (link). Mice were completely anesthetized with avertin using 300 μl per mouse with tert-amyl alcohol content at 2.5% and 2,2,2 tribromoethanol (TCI America, T1420) at a concentration of 50 mg per kg. Soluble OVA, apoptotic cells and TLR agonists were delivered in a 50 μl volume. Final concentration of deliveries were 1 μg soluble OVA (sOVA) (0.22 micron filtered, Grade VII Sigma) or 20 × 106 apoptotic cells ± 50μg R848 (Enzo Life Sciences) or ± 10 μg Poly I:C (Enzo Life Sciences). Mice were sacrificed at various times depending on the experimental design.
Desch A.N., Gibbings S.L., Clambey E.T., Janssen W.J., Slansky J.E., Kedl R.M., Henson P.M, & Jakubzick C. (2014). Dendritic cell subsets require cis-activation for cytotoxic CD8 T cell induction. Nature communications, 5, 4674.
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10

OVA and Poly I:C Antigen Presentation

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Mice were delivered were 100 μg soluble OVA (sOVA) (0.22 micron filtered, Grade VII Sigma) ± 100 μg Poly I:C (Enzo Life Sciences). Four days later, LLN were harvested for H-2Kb/SIINFEKL tetramer staining (Beckman Coulter) for flow analysis.
Desch A.N., Gibbings S.L., Clambey E.T., Janssen W.J., Slansky J.E., Kedl R.M., Henson P.M, & Jakubzick C. (2014). Dendritic cell subsets require cis-activation for cytotoxic CD8 T cell induction. Nature communications, 5, 4674.
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