The human pulmonary mucoepidermoid carcinoma cell line NCI-H292 (ATCC CRL-1848) was maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37C in a humidified atmosphere with 5% (v/v) CO2. The NCI-H292 cells were expanded in 24-well plates and maintained in serum-free RPMI 1640 medium for 6 h before stimulation with mithramycin A (Cayman Chemical, Ann Arbor, MI, USA) for 30 min or with ML-60218 (Cayman Chemical) for 2 h. Subsequently, the cells were transfected with 0.5 µg of poly(dA:dT; Thermo Fisher Scientific) using polyethylenimine MAX (Polysciences, Warrington, FL, USA) and incubated for 6 h. The transfected cells were subsequently exposed to 25 µg/mL poly(I: C; Sigma-Aldrich, St. Louis, MO, USA) and 4 ng/mL TGF-α (R&D Systems, Minneapolis, MN, USA) for 6 h at 37C. For plasmid stimulation, the cells were transfected with 0.5 µg of pcDNA6.2-EGFP in 24-well plates, using FuGENE HD (Promega, Madison, WI, USA). For cGAMP (Thermo Fisher Scientific)-mediated cellular stimulation, cells were incubated in the presence of cGAMP for 16 h.
Cgamp
Manufactured by Thermo Fisher Scientific
Sourced in Japan, United States
The CGAMP is a lab equipment product designed for the analysis and quantification of cyclic guanosine monophosphate (cGMP) levels in various biological samples. It provides a reliable and efficient method for researchers to measure cGMP, which is an important second messenger molecule involved in various physiological processes.
Automatically generated - may contain errors
Lab products found in correlation
Poly da dt, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Mithramycin a, by Cayman Chemical (1 mentions)
Tgf α, by R&D Systems (1 mentions)
Ml 60218, by Cayman Chemical (1 mentions)
Polyethylenimine max, by Polysciences (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
Lyovec, by InvivoGen (1 mentions)
Iscove s modified dulbecco s media, by Thermo Fisher Scientific (1 mentions)
Anti cd3 and anti cd28, by BioLegend (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Diabzi, by Cayman Chemical (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
5 ppprna, by InvivoGen (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Cgamp, by InvivoGen (1 mentions)
Herring testes dna ht dna, by Merck Group (1 mentions)
7 protocols using cgamp
1
Mucoepidermoid Carcinoma Cell Stimulation
2
HSV-2 Stimulation and cGAMP Delivery
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For in vitro HSV-2 stimulation, multiplicity of infection (MOI) at 0.5 or 1 was used.
For transfection setups, 4 µg/mL dsDNA (HSV-60; InvivoGen) and 4 µL/mL Lipofectamine2000 (Invitrogen) were used according to the manufacturer’s instructions. Furthermore, cGAMP (Invitrogen) was used at a concentration of 4 µg/mL together 4 µL/mL Lipofectamine2000. Stimulation with cGAMP was performed using 4 µg/mL delivered to cells using Lipofectacmine2000 (Invitrogen).
For transfection setups, 4 µg/mL dsDNA (HSV-60; InvivoGen) and 4 µL/mL Lipofectamine2000 (Invitrogen) were used according to the manufacturer’s instructions. Furthermore, cGAMP (Invitrogen) was used at a concentration of 4 µg/mL together 4 µL/mL Lipofectamine2000. Stimulation with cGAMP was performed using 4 µg/mL delivered to cells using Lipofectacmine2000 (Invitrogen).
3
HDM-Induced Allergic Asthma Model
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HDM extract (from Dermatophagoides farinae); LPS: 20200 EU/mg and protease: 0.205 Relative activity (0.5 μg HDM/0.01U papain) was kindly provided by the Institute of Tokyo Environmental Allergy (ITEA), Inc. (Tokyo, Japan). Allergic asthma-induced mouse models have already been established, and some modifications have been made to a method that can reproduce the pathophysiology of asthma56 (link),58 (link),59 (link). C57BL/6N (WT) or Sting−/− mice were anesthetized by intraperitoneally administering ketamine (90 mg/kg) and medetomidine (1 mg/kg), following which we intratracheally injected them with 30 µg of HDM or normal saline (Ootsuka, Tokushima, Japan) once every other day, for a total of seven times. Twenty-four hours after the final injection, the HDM-sensitized mice were sacrificed and analyzed. In some experiments, 10 µg cGAMP (Thermo Fisher Scientific, Tokyo, Japan) was administered in addition to HDM or saline.
4
Stimulating T Cell Activation via DCs
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DCs were infected with SL3261 murine cyclic GMP–AMP synthetase (mcGAS) or SL3261 mcGASAA for 30 min, spun down, and the medium was replaced with RPMI-1640 supplemented with 10% FCS and 100 µg/mL gentamycin. Next, the stimuli LPS or cGAMP (10 µg/mL, Invitrogen) were added to the corresponding wells. The cGAMPs were incubated with transfection reagent Lyovec (Invivogen) according to manufacturer’s instructions to ensure intracellular delivery of the cGAMPs prior to adding to DCs. After 16-hour incubation, DCs were cocultured with allogeneic PBLs in Iscove’s Modified Dulbecco’s Media (Gibco) supplemented with 10% FCS and 1% penicillin/streptomycin, using a 1:8 ratio. As a positive control, T cells were stimulated with plate-bound anti-CD3 and anti-CD28 (BioLegend). After 3 days, IL-2 (20 U/mL, Chiron) was added to the cocultures. After 6 days, PBLs were harvested, plated in 96-well plates, and incubated for 4 hours with brefeldin A (10 µg/mL, Sigma), after which they were analyzed for cytokine production by flow cytometry as described further.
5
Intratracheal STING Agonist Challenge in Mice
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Mice were anesthetized with 2% Isoflurane (ISO-VET, Netherlands) and challenged intratracheally (i.t) with cGAMP (1–10 µg in 40 µL, Invitrogen, California) or diABZI, a STING agonist 3 (0.01 to 1 µg in 40 µL, Cayman chemicals, Michigan) or saline (NaCl 0.9%) for 3 consecutive days. Bronchoalveolar lavage (BAL) was performed 24 h after the last challenge by flushing lung tissue four times with 0.5 mL of cold NaCl 0.9% via a cannula inserted in the trachea. BALF was collected, cells counted and cytospins performed. The supernatant of the first lavage was collected after centrifugation and stored at −80 °C for dsDNA quantification. The left lung lobe was harvested for histology, the post caval lung for RNA extraction and qPCR analysis, and the right lobes for Western blots analysis and cytokines measurement. Protein extravasation in the BALF was measured by Pierce™ BCA Protein Assay (ThermoFisher®, Massachusetts).
6
Viral Infection and Immune Stimulation Assay
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HSV-1 was a gift from S. Paludan (Aarhus University, Denmark). MVA was a gift from I. Drexler (Düsseldorf University, Germany). SeV Cantell strain was from ECACC. Serial dilutions of SeV indicated the optimal dilution to elicit IFN without causing cell death as 1:200, which was used in all experiments. All other viruses were used at a multiplicity of infection of 10. Cell stimulants used were 70-mer dsDNA (70-mer) (9 (link)); poly(dA-dT), poly(I:C) (both Sigma-Aldrich); cGAMP and 5′pppRNA (both InvivoGen). 70-mer, poly(dA-dT), poly(I:C), 5′pppRNA, and cGAMP were delivered to cells by transfection using Lipofectamine®2000 (Invitrogen), at the concentrations indicated in figure legends. IL-1α (PBL Interferon Source) and TNFα (PeproTech) were also used.
7
Transfection of Diverse Nucleic Acids
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Transfection of siRNA, cGAMP, mtDNA, herring testis (HT), or plasmid DNA was performed using RNAiMax according to the maunfacturer's instructions (Invitrogen). mtDNA was isolated from mitochondria using the Mitochondrial Isolation Kit for Mammalian cells (Thermo Fisher Scientific) and mtDNA was purified by QIAamp® DNA Mini Kit. Mouse mtDNA was prepared from PolgA+/+ MEFs and human mtDNA was prepared from primary human skin fibroblasts. Herring Testes DNA (HT‐DNA) was purchased from Sigma‐Aldrich (D6898).
Mouse mtDNA, human mtDNA, or mtDNA that had been digested by 20 μg/ml DNase I (Sigma‐Aldrich) for 30 min at 37°C as DNase I‐pretreated mtDNA, were transfected with RNAiMax for 48 h. 4 μg/ml cGAMP (Sigma‐Aldrich, 5.31889.0001) was transfected with RNAiMax for 24 h. Mouse mtDNA was transfected at a concentration of 100 ng/ml into MEFs or pmAT2. Human mtDNA was used at a concentration of 200 ng/ml for phLF transfection. siRNAs were transfected for 48 h at a final concentration of 5 nM. Details on the siRNAs applied in this study are provided in TableEV4 . HT‐DNA or plasmid DNA was transfected at a concentration of 1 μg/ml.
Mouse mtDNA, human mtDNA, or mtDNA that had been digested by 20 μg/ml DNase I (Sigma‐Aldrich) for 30 min at 37°C as DNase I‐pretreated mtDNA, were transfected with RNAiMax for 48 h. 4 μg/ml cGAMP (Sigma‐Aldrich, 5.31889.0001) was transfected with RNAiMax for 24 h. Mouse mtDNA was transfected at a concentration of 100 ng/ml into MEFs or pmAT2. Human mtDNA was used at a concentration of 200 ng/ml for phLF transfection. siRNAs were transfected for 48 h at a final concentration of 5 nM. Details on the siRNAs applied in this study are provided in Table
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