MC38-luc orthotopic tumor-bearing mice were injected with 10 μg/mouse 2’3’-Cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP; Millipore Sigma) intratumorally on day eight. Mice received SCRT, and immediately following each dose of RT were intravenously injected in the tail vein with 20μg cGAMP (in PBS). Tumor burden was monitored by IVIS or mice sacrificed on day twenty-six and primary tumors weighed.
Cgamp
Manufactured by Merck Group
CGAMP is a laboratory equipment product manufactured by Merck Group. It is a device used for the detection and quantification of cyclic guanosine monophosphate (cGMP), a signaling molecule involved in various biological processes. The core function of CGAMP is to enable researchers and scientists to measure cGMP levels in their experiments and studies.
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Lab products found in correlation
C di gmp, by Merck Group (2 mentions)
Cgamp, by Thermo Fisher Scientific (1 mentions)
Herring testes dna ht dna, by Merck Group (1 mentions)
Mitochondrial isolation kit for mammalian cells, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Merck Group (1 mentions)
Qiaamp dna mini kit, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Poly 1 c, by Bio-Techne (1 mentions)
Cgamp, by InvivoGen (1 mentions)
Mg132, by MedChemExpress (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ap20187, by Takara Bio (1 mentions)
Bafa1, by Selleck Chemicals (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Diabzi, by Selleck Chemicals (1 mentions)
Dmxaa, by Bio-Techne (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
7 protocols using cgamp
1
Enhancing Tumor Immunotherapy with cGAMP
2
Combining cGAMP and SCRT for Tumor Regression
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MC38-luc orthotopic tumor-bearing mice were injected with 10 µg/mouse 2’3’-Cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP; Millipore Sigma) intratumorally on day eight. Mice received SCRT, and immediately following each dose of RT were intravenously injected in the tail vein with 20 µg cGAMP (in PBS). Tumor burden was monitored by IVIS or mice sacrificed on day twenty-six and primary tumors weighed.
3
Synthesis and Purification of Labeled RNA Hairpins
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All RNAs were synthesized by run-off in vitro transcription using T7 RNA polymerase as previously described (Stoldt et al. 1998 (link)). Unlabeled, 15N-adenine or 13C,15N-adenine labeled Vc2, Vc2 G20A, Gs1761, Gs1761WT as well as Cbe 1–2 were transcribed using SmaI linearized plasmid DNA or PCR generated double-stranded DNA fragments as templates and purified by denaturing PAGE as previously described (Duchardt-Ferner et al. 2010 (link)). 13C,15N- and 15N-labeled rNTPs were purchased commercially (Silantes).
RNAs were folded under conditions favoring monomeric hairpin structures in a low salt buffer (2.5 mM Bis-Tris, pH 6.5) by denaturing at 70°C for 5 min, rapid 10-fold dilution with the same ice-cold buffer and subsequent annealing on ice. They were exchanged into 25 mM Bis-Tris buffer, pH 6.5, 5 mM magnesium acetate for NMR spectroscopy using ultracentrifugation devices (VivaSpin 2, MWC 3 kDa) and multiple cycles of concentration and dilution with NMR buffer. The RNAs in the final NMR samples were monomeric and homogeneous as judged from a native gel (Supplemental Fig. S5 ).
Unlabeled c-di-GMP and c-GAMP were purchased commercially as sodium salts (Sigma Aldrich) and dissolved in NMR buffer.
RNAs were folded under conditions favoring monomeric hairpin structures in a low salt buffer (2.5 mM Bis-Tris, pH 6.5) by denaturing at 70°C for 5 min, rapid 10-fold dilution with the same ice-cold buffer and subsequent annealing on ice. They were exchanged into 25 mM Bis-Tris buffer, pH 6.5, 5 mM magnesium acetate for NMR spectroscopy using ultracentrifugation devices (VivaSpin 2, MWC 3 kDa) and multiple cycles of concentration and dilution with NMR buffer. The RNAs in the final NMR samples were monomeric and homogeneous as judged from a native gel (
Unlabeled c-di-GMP and c-GAMP were purchased commercially as sodium salts (Sigma Aldrich) and dissolved in NMR buffer.
4
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
5
Stimulation of Innate Immune Pathways
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Poly(I:C) (Tocris) dissolved in 0.9% NaCl was introduced into HeLa cells using Lipofectamine 2000 at 500 ng/mL for 6 h. cGAMP (Invivogen) was delivered into cells either by direct treatment at the indicated concentrations and durations or by digitonin permeabilization.78 (link) For digitonin permeabilization, cells were incubated with the permeabilization buffer (50 mM HEPES, 100 mM KCl, 3 mM MgCl2, 0.1 mM DTT, 85 mM sucrose, 0.2% BSA, 1 mM ATP, 0.1 mM GTP, and 10 μg/mL digitonin) with or without cGAMP at 4 μM for 30 min at 37°C. The permeabilization buffer was then replaced with fresh DMEM, and the cells were further incubated at 37°C for 3 h before harvest. Interferon-β (Bio-Techne) was dissolved in 10 mM citric acid, 1% BSA, and 0.5% CHAPS. Dissolved interferon-β was diluted in DMEM at 10 ng/mL for direct treatment for 24 h. MG132 (MedChemExpress) treatment was carried out at 20 μM for 12 h. BMS-345541 (Adooq Bioscience) treatment was performed at 50 μM for 1 h prior to and during Poly(I:C) or cGAMP stimulation, or for a total of 7 h for rescue experiments. For chloroquine (Sigma-Aldrich) treatment, cells were incubated with the drug at 50 μM for 30 min prior to cGAMP stimulation, during the stimulation, and during the post-stimulation incubation for a total of 4 h.
6
Cell Culture and Transfection Protocol
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L929 and HEK293T cells were purchased from ATCC (American Type Culture Collection) and BJ cells was a kindly gift from Dr. Daxing Gao of University of Science and Technology of China (USTC), and all the above cells were maintained in DMEM (Gibco) with 10 % fetal bovine serum (FBS, Hyclone) and 100 units/ml penicillin (Gibco) plus 100 μg/ml streptomycin (Gibco). Plasmids were purified with endotoxin-free purification kits (CoWin Biosciences, CW2105) and transfected into cells with Lipofectamine 3000 (Invitrogen) according to the user manual. For small molecule inhibitors: DMXAA (Tocris Bioscience, #5601), EACC (MedChemExpress, HY-129111), diABZI (Selleck, S8796), AP20187 (Takara, #635058), Baf A1 (Selleck, S1413), cGAMP (Sigma, SML1229).
7
HPLC for c-di-AMP Kinetics and Quantification
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