Poly da dt lyovec

Manufactured by InvivoGen

Sourced in United States

Poly(dA:dT)/LyoVec is a transfection reagent. It is composed of a synthetic double-stranded DNA, poly(dA:dT), complexed with the cationic lipid LyoVec. This complex facilitates the delivery of nucleic acids into mammalian cells.

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15 protocols using poly da dt lyovec

Pathogen Recognition Receptor Agonists

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Pathogen recognition receptor (PRR) agonists lipopeptides Pam2csk4, FSC-1, Poly(I:C) HMW/naked, Poly(I:C) LMW/naked, HKLM, Poly(dA:dT)/LyoVec, LPS, Imiquimod, CpG (ODN2006), FLA, ss40, Poly(I:C)HMW/LyoVec and Poly(I:C)LMW/LyoVec were purchased from InvivoGen (San Diego, CA). Human recombinant cytokines of IL36α, IL36β, IL36γ, IL1α, IL1β, TNFα, IL18, IL33, IL17A, IL-4, IL13, IFN-κ and IFN-γ were purchased from R&D Biosystems.

Li Y., Song Y., Zhu L., Wang X., Yang B., Lu P., Chen Q., Bin L, & Deng L. (2019). Interferon Kappa Is Up-Regulated in Psoriasis and It Up-Regulates Psoriasis-Associated Cytokines in vivo. Clinical, Cosmetic and Investigational Dermatology, 12, 865-873.

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Inflammasome Activation in iPS-Derived Cells

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iPS-MLs or iPS-MPs were harvested as described above and seeded into 96-well plates at 5 × 10⁴ cells per well. When indicated, cells were pretreated with ABT-751 (10 µM; Sigma-Aldrich), CA4P (10 µM; Sigma-Aldrich), MCC950 (10 µM; Sigma-Aldrich), and colchicine (100 ng/ml; Sigma-Aldrich) 30 min before LPS priming or with 2BP (20 µM; Sigma-Aldrich) and bryostatin (0.1 µM; Sigma-Aldrich) 24 h before harvest. For inflammasome activation, cells were primed with LPS (1 µg/ml) for 4 h and treated with UCN-01 (10 µM; Sigma-Aldrich), nigericin (10 µM; Sigma-Aldrich), or purified flagellin from Salmonella typhimurium (InvivoGen) in DOTAP liposomes (Roche; IC-FLA; 6 µg DOTAP/1 µg FLA-ST) for an additional 2 h, after which the supernatants were collected. For TcdA stimulation, TcdA (1 µg/ml; List Biological Laboratories) was added after 2 h of LPS priming (1 µg/ml) and the supernatants were collected 4 h later. For AIM2 inflammasome activation, poly (dA; dT)/Lyovec (1 µg/ml; InvivoGen) was added along with LPS priming (1 µg/ml), and the supernatants were collected 4 h later. The IL-1β, IL-18, and IL-6 concentrations were measured using the Bio-Plex Pro Human Cytokine Assay (BioRad).

Nishitani-Isa M., Mukai K., Honda Y., Nihira H., Tanaka T., Shibata H., Kodama K., Hiejima E., Izawa K., Kawasaki Y., Osawa M., Katata Y., Onodera S., Watanabe T., Uchida T., Kure S., Takita J., Ohara O., Saito M.K., Nishikomori R., Taguchi T., Sasahara Y, & Yasumi T. (2022). Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation. The Journal of Experimental Medicine, 219(6), e20211889.

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CRISPR-Mediated Gene Editing Workflow

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LentiCRISPRv2-puro, psPAX2, and pMD2.G plasmids were provided by Dr. Jian Huang (Department of Pathology and Laboratory Medicine, Temple University, USA). PE Annexin V Apoptosis Detection Kit I was purchased from BD (Pharmingen, USA). Poly(dA:dT), LyoVec, and puromycin were purchased from InvivoGen (San Diego, CA, USA). Lipofectamine 3000 Reagent was purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Antibodies against RIG-I, DNA sensors, ISGs, signal transducers and activators of transcription (STATs), and IRFs were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against HSV-1+HSV-2 gD, HSV-1+HSV-2 gB were purchased from Abcam (Cambridge, UK). Antibody to GAPDH was purchased from Proteintech (Chicago, USA).

Shao D.D., Meng F.Z., Liu Y., Xu X.Q., Wang X., Hu W.H., Hou W, & Ho W.Z. (2021). Poly(dA:dT) Suppresses HSV-2 Infection of Human Cervical Epithelial Cells Through RIG-I Activation. Frontiers in Immunology, 11, 598884.

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Primed THP-1 Cells Inflammasome Activation

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Primed THP-1 cells were treated for 45 min with the indicated inhibitor in Opti-MEM medium (Life Technology) prior to activation by 5 mM ATP, 1 μM nigericin, 500 μg/ml MSU crystals, 5 μg/ml Poly(dA:dT)/LyoVec™ (Invivogen) or 3 μg FLA-PA (transfected with ViaFect) for 6 h (MSU crystals were a kind gift of Dr. A. Scanu, University of Padova). Culture supernatants were tested for the production of IL-1β or caspase-1 by enzyme immunoassay (Human IL-1β, eBioscience, San Diego, CA, USA and Caspase-1, Quantikine; R&D Systems, Minneapolis, MN, USA).

Burger D., Fickentscher C., de Moerloose P, & Brandt K.J. (2016). F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2. Scientific Reports, 6, 29834.

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Phagocytosis of Mitochondria-Enriched Red Blood Cells

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Mito⁺ and Mito⁻ RBCs were generated from HD PBMCs in the presence or absence of MG132 (5 μM) respectively. To deplete mtDNA, 2′,3′-Dideoxycytidine (ddC, 100 μM; Sigma–Aldrich) was added during the culture together with MG132. RBCs were then opsonized with anti-human RBC antibody (diluted 1:250; Rockland), stained with 2 μM CFSE (Thermo-Fisher) or with 100 ng/mL pHrodo™ Red SE (Thermo-Fisher) and then co-cultured with macrophages at a 1:2 (M⏀ to RBCs ratio) for 18 h in cRPMI medium with 10% FBS. For some experiments, RBCs were oxidized with Copper (II) sulfate (0.5 mM; Santa Cruz Biotech) and Sodium L-ascorbate (100 mM; Santa Cruz Biotech) or aged with Calcium Chloride (2.5 mM; Santa Cruz Biotech) and A23187 (0.5 μM; Sigma–Aldrich) for 30 min at 37°C. The supernatants were collected and frozen for the quantification of cytokine levels with the CBA assay (BD Bioscience). Non-engulfed RBCs were removed by three washes with cold PBS, phagocytes were collected using trypsin and EDTA and assessed for phagocytosis by flow cytometry or immunofluorescence microscopy upon counterstaining the actin filaments with AlexaFluor 647 Phalloidin (Thermo-Fisher) and nuclear DNA with Hoechst 33342 (Thermo-Fisher). Pre-complexed Poly dA:dT/ LyoVec™ (referred as Poly dA:dT. 1 μg/mL; Invivogen) was used as a positive control.

Caielli S., Cardenas J., de Jesus A.A., Baisch J., Walters L., Blanck J.P., Balasubramanian P., Stagnar C., Ohouo M., Hong S., Nassi L., Stewart K., Fuller J., Gu J., Banchereau J.F., Wright T., Goldbach-Mansky R, & Pascual V. (2021). ERYTHROID MITOCHONDRIAL RETENTION TRIGGERS MYELOID-DEPENDENT TYPE I INTERFERON IN HUMAN SLE. Cell, 184(17), 4464-4479.e19.

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Monitoring Immune Response Signaling Pathways

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RNA polymerase subunit C32 and SRSF1 antibodies were purchased from Santa Cruz Biotechnology: Pol III RPC32 antibody (H-9): sc-48365, SF2/ASF Antibody (P-15): sc-10254. Poly (dA:dT)/LyoVec, poly (I:C)/LyoVec, and 5′ppp-dsRNA were purchased from Invivogen. Human IFN-β ELISA kits were obtained from PBL Interferon Source. Human IL-6, TNFα, and IL-1β ELISA Duoset were purchased from R&D Systems. ML-60218 was purchased from SYMANSIS.

Xue F., Li X., Zhao X., Wang L., Liu M., Shi R, & Zheng J. (2015). SRSF1 Facilitates Cytosolic DNA-Induced Production of Type I Interferons Recognized by RIG-I. PLoS ONE, 10(2), e0115354.

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Evaluating THP-1 and HEK-Blue Responses to Immune Stimuli

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THP-1 cells were seeded in 96-well plates (Corning Costar) at a density of 100,000 cells per well. First, cells were stimulated for 18 h with rSIP, FLH, LPS, a NOD 1 ligand (positive control for NF-kB; C12-iE-DAP, InvivoGen), and a PRR agonist (positive control for IRF; Poly (dA:dT)/LyoVec™, InvivoGen). The supernatant was then subjected to a colorimetric enzyme assay to measure alkaline phosphatase (AP) activity using the commercial QUANTI-Blue™ solution (InvivoGen). The supernatant was then incubated at 37°C for 3 h, and the optical density was read at 650 nm in an Epoch 2 reader (BioTek). On the other hand, luciferase activity (LUCIA) was measured using the commercial solution QUANTI-Luc ™ (InvivoGen), which has a coelenterazine substrate and stabilizing agents for the luciferase reaction. The light signal produced was then quantified using a Berthold luminometer (Model LB9515), and the signal was expressed as relative light units (RLUs).
Hek-Blue cells (hkb-mtlr4 and hkb-htlr4, InvivoGen) express SEAP under the control of promoters containing binding elements for the NF-κB transcription factor (35 (link)). Hek-Blue cells were seeded in 96-well plates (Corning Costar) at a density of 25,000 cells per well in HEK-Blue™ Detection medium (InvivoGen). Then, the cells were stimulated for 48 h with rSIP, FLH, and LPS, and SEAP was quantified using an Epoch 2 reader (Biotek).

Díaz-Dinamarca D.A., Salazar M.L., Escobar D.F., Castillo B.N., Valdebenito B., Díaz P., Manubens A., Salazar F., Troncoso M.F., Lavandero S., Díaz J., Becker M.I, & Vásquez A.E. (2023). Surface immunogenic protein from Streptococcus agalactiae and Fissurella latimarginata hemocyanin are TLR4 ligands and activate MyD88- and TRIF dependent signaling pathways. Frontiers in Immunology, 14, 1186188.

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Inflammasome Activation Pathway Analysis

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Anti-IL-1β antibodies (AF-401-NA) were from RD Systems, anti-GSDMD (ab209845) and anti-NEK7 antibodies (ab133514) were from Abcam, anti-caspase-1 (AG-20B-0042B) and anti-ASC antibodies (AG-25B-0006) were from Adipogen, and the anti-αtubulin antibodies (ARG65693) were from Arigo. LPS (Escherichia coli O111:B4; #L2630), ATP (A6419), and nigericin (481990) were obtained from Sigma. LND (AF-1890) and imiquimod (R-837) were obtained from Selleck. MSU crystals (tlrl-msu), muramyldipeptide (tlrl-mdp), flagellin from S. typhimurium (tlrl-stfla) and poly(dA:dT)/LyoVec™ were from Invivogen. Anti-hexokinase 2 antibodies (NBP2-02272) were from Novus. Anti-NLRP3 antibodies (15101) were from Cell Signaling Technology. Goat anti-rabbit IgG (HRP) (arg65351), donkey anti-goat IgG (HRP) (arg65352) and goat anti-mouse IgG antibodies (HRP) (arg65350) were from Arigo. Disuccinimidyl suberate (DSS) was purchased from Thermo Scientific.

Chen C., Zhou Y., Ning X., Li S., Xue D., Wei C., Zhu Z., Sheng L., Lu B., Li Y., Ye X., Fu Y., Bai C., Cai W., Ding Y., Lin S., Yan G., Huang Y, & Yin W. (2022). Directly targeting ASC by lonidamine alleviates inflammasome-driven diseases. Journal of Neuroinflammation, 19, 315.

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Cell Culture and Infection Protocols

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HeLa cells, WT (wild-type) and TREX1 KO mouse embryonic fibroblasts (MEFs), and HEK293T were cultured in supplemented DMEM and mouse J774 cells cultured in complete media for macrophages, as described earlier (25 (link)). Mouse BM1.11 cells (26 (link)) and human OE-E6/E7 (oviduct epithelial cells) (27 (link)) were cultured in supplemented F12-DMEM as described (25 (link)). Poly dA:dT/LyoVec (100 µg/ml), Poly I:C/LyoVec (50 µg/ml) and 2’3’ cGAMP (1 mg/ml) were purchased from Invivogen. Carbenoxolone (working concentration 0.2 mM) was purchased from SIGMA. pBluscript vector (1–2.5 µg) was used as immunostimulatory DNA (ISD). cDNA constructs for human cGAS (pCMV-cGAS) and STING (pCMV-STING) were purchased from Origene. C. muridarum, C. trachomatis D and L2 were propagated in McCoy cells and infections performed at 1 MOI or as indicated, as previously described (25 (link)).

Zhang Y., Yeruva L., Marinov A., Prantner D., Wyrick P., Lupashin V, & Nagarajan U.M. (2014). The DNA sensor, cyclic GMP-AMP synthase (cGAS) is essential for induction of IFN beta during Chlamydia trachomatis infection. Journal of immunology (Baltimore, Md. : 1950), 193(5), 2394-2404.

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Liposomal Transfection and Immune Stimulants

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DOTAP liposomal transfection reagent was from Roche (Mannheim, Germany). Cholera toxin B subunit (CTB) was from List Biological Laboratories, Inc. Ultra-pure lipopolysaccharide (E. coli O111:B4), ultra-pure lipopolysaccharide (S. minnesota RE595), poly(I:C) LMW and poly(dA:dT)/lyovec were from Invivogen. ATP, apyrase, carbenoxolone (CBX), bafliomycin A, brefeldin (BFA), 18-glycyrrhetinic (18GA), flufenamic acid (FFA), glibenclamide, gadolinium III (Gd3), probenecid, ARL67156 (an ecto-ATPase inhibitor) and trovafloxacin (a pannexin-1-selective antagonist) were from Sigma-Aldrich. Alum was from Thermo Scientific. Nigericin and Ac-DNLD-CHO (caspase-3/7 inhibitor) were from Calbiochem, and zVAD-FMK (pan-caspase-inhibitor) and zDEVD-FMK (caspase-3 inhibitor) was from R&D system. Fluorescent Yo-Pro-1 was from Life Technology.

Yang D., He Y., Muñoz-Planillo R., Liu Q, & Núñez G. (2015). Caspase-11 requires the pannexin-1 channel and the purinergic P2X7 pore to mediate pyroptosis and endotoxic shock. Immunity, 43(5), 923-932.

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