ISD017 and derived peptides were obtained from Schafer-N. H-151 and RU.521 were obtained from InvivoGen. To dissolve ISD017 a panel of solvents were tested, namely: H2O; 0.9% NaCl; TBS (pH 7.3); PBS (pH 7.4); HEPES; PBS (pH7.4) + 1 M NaOH. For the latter, which was most successful, 194 uL of PBS mixed with 6 uL 1 M NaOH was added to 1 mg ISD017 or mutant peptides. H-151 was dissolved in DMSO and diluted in PBS + 10% tween80, and RU.521 was dissolved in DMSO. For in vitro experiments, primary cells and cell lines were treated with cGAS/STING antagonists one h prior to stimulation unless otherwise stated. The antagonists were administered by direct addition to the culture medium. For stimulations, we used 60‐mer dsDNA (DNA technology) [47] (link), 2′3′ cGAMP, and poly(I:C) (both from InvivoGen). The agonists were delivered with Lipofectamine 2000 (ThermoFischer). Liposomes were prepared as described using lipid blends containing DOTAP, DOPE and lissamine–rhodamine DOPE in the w/w/w ratio 1/1/0.1 Avanti Polar Lipids [48] (link).
Ru 521
Manufactured by InvivoGen
RU.521 is a laboratory equipment product. It is a device used for scientific research and experimentation. The core function of RU.521 is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
H 151, by InvivoGen (4 mentions)
Inh ru521, by InvivoGen (4 mentions)
2 3 cgamp, by InvivoGen (3 mentions)
Bx795, by InvivoGen (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
G3 ysd, by InvivoGen (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Bafilomycin, by InvivoGen (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Itacitinib, by MedChemExpress (2 mentions)
Odn20959, by Miltenyi Biotec (2 mentions)
Mrt67037, by InvivoGen (2 mentions)
Pam3csk4, by InvivoGen (2 mentions)
Ruxolitinib, by MedChemExpress (2 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Lyovec, by InvivoGen (1 mentions)
Tlrl nacga23 02, by InvivoGen (1 mentions)
Lyec 12, by InvivoGen (1 mentions)
Tlrl bx7, by InvivoGen (1 mentions)
Pik93, by Merck Group (1 mentions)
18 protocols using ru 521
1
Solvents and Antagonists for cGAS/STING Studies
2
Characterizing 2'3'-cGAMP Signaling Pathway
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2′3′-cGAMP was purchased from Invivogen. 2′3′-cGAMP (5 µg/ml) (Invivogen, tlrl-nacga23-02) was added to the cells complexed with LyoVec (Invivogen, Lyec-12) in order to aid internalization. ISD (Interferon stimulatory DNA)/LyoVec (Invitrogen, tlrl-isdc, 1 µg) was also used. Membrane lipid strips were obtained from Echelon. The following inhibitors were used: Fura-2 AM, Ca2+ selective fluorescent indicator (Abcam, ab120873), BX795 (TBK-1 inhibitor, Invivogen, tlrl-bx7, 1 µM), RU.521 (cGAS inhibitor, Invivogen, inh-ru521, 500 nM). GSK2998533 (100 nM) & GSK2683449 (100 nM) were provided by GlaxoSmithKline (GSK). PIK93 was also used (Sigma Aldrich, 1 µM)
3
Modulating cGAS-STING Signaling in Cells
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Cells were treated with a specific concentration of 2′3′-cGAMP (Invivogen) or cGAS inhibitor Ru521 (Invivogen) in digitonin permeabilization buffer (50 mM HEPES [pH 7.0], 100 mM KCl, 85 mM sucrose, 3 mM MgCl2, 0.2% bovine serum albumin [BSA], 1 mM ATP, 0.1 mM DTT, and 10 μg/ml digitonin) (28 (link)). The cells were incubated for 30 min at 37°C, and then the cells were washed with phosphate-buffered saline (PBS), and fresh medium or virus inoculation was added as indicated. For the Ru521-treated samples, cells were incubated for 2 h with fresh DMEM before being infected.
4
Murine Bone Marrow Dendritic Cell Differentiation and Stimulation
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Bone marrow was harvested from 8-10 week-old C57BL/6J and STING gt/gt mice. RBC cells were lysed and cells were plated at a density of 106/mL at 37°C in RPMI-1640 containing 2 mM L-glutamine, 10% (v/v) heat inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 25 ng/mL murine GM-CSF (PeproTech). On days 3 and 5 cells were lifted from the plates using gentle scraping and counted alongside cells in suspension and replated at 106/mL. On Day 7 cells were harvested for in vitro experiments. Cells were cultured at a density of 0.5x106/mL in the appropriate medium in a 12 well plate. Twenty-four hours post culture initiation, the medium was replaced with fresh medium or medium containing various combinations of: 2.5 µg/mL ONP-302, 8 µg/mL RU521 (STING inhibitor; In vivogen), 20 µM Z-VAD-FMK (Caspase inhibitor; In vivogen), 400 ng/mL G3-YSD (cDNA; In vivogen), and/or 1 μg/mL LPS. After 18-24 hours, cell supernatants were collected and frozen at -20°C for use in Milliplex assays.
5
Immune Response Modulation Protocol
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PAM3CSK4 was purchased from Invivogen and was reconstituted in endotoxin‐free H2O. IFN‐α and IFN‐β were purchased from PBL Interferon Source. B18R was purchased from R&D Systems. TNF‐α was purchased from PeproTech. PMA (Sigma), TLR3 inhibitor (Calbiochem), TLR7/8 inhibitor ODN20959 (Miltenyi), A151 (Invivogen), ruxolitinib (MedChemExpress), itacitinib (MedChemExpress), bafilomycin (Invivogen), BX795 (Invivogen), MRT67037 (Invivogen), H‐151 (Invivogen), G150 (Selleckchem), and RU.521 (Invivogen) were reconstituted in DMSO or sterile deionized water.
6
Inhibition of Inflammatory Pathways
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For specific experiments, cells were treated with 0.1, 1, or 10 µM of the NLRP3 inhibitor, MCC950 (InvivoGen), or the caspase-1 inhibitor, VX-760 (InvivoGen); 10 or 20 µM of the cGAS inhibitor, RU.521 (InvivoGen); 25 µg mL–1 of the K+ channel inhibitor, Glybenclamide (InvivoGen); 10 µM of the cathepsin B inhibitor, ZRLR (kindly provided by Eva Wieczerzak68 (link)); 5 µM of the phagocytosis inhibitor, cytochalasin D (Sigma-Aldrich); or with 5 mM of ATP (InvivoGen); 1 µg mL–1 of the RIG-I agonist, 5’ppp-dsRNA (InvivoGen).
7
Inhibition of Arp2/3 and cGAS-STING Pathways
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Cells were treated with 25 μM of Arp2/3 inhibitor CK666 (Tocris Bioscience, 3950), Lat-A (0.5 μM; MilliporeSigma, L5163). Control cells were treated with an equivalent concentration of DMSO. After 30 minutes of incubation, cells were washed and used for the different experiments. The STING inhibitors C-176 (PC-35311) and C-178 (PC-35310) (34 (link)) were purchased from ProbeChem. The cGAS inhibitor RU.521 was purchased from Invivogen (inh-ru521). For in vitro treatments, cells were preincubated with 0.5 μM C-176, 50 μM RU.521, or the equivalent concentration of DMSO. After 30 minutes, stimuli were added to the wells without washing the inhibitor. RNA or cell culture supernatant was collected after 3 and 16 hours, respectively. For in vivo treatment, mice were injected with 7.5 μL C-176 or DMSO dissolved in 85 μL corn oil twice per day for 11 consecutive days. Mice were euthanized, and total spleen was harvested. RNA was extracted from single-cell suspension to perform RT-PCR.
8
Quantifying Cellular Antiviral Response
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THP1-Lucia ISG cells (Invivogen) were seeded into a 96-well plate and incubated with RU.521 (Invivogen) or XQ2 for the indicated time, and then transfected with dsDNA by lipofectamine 2000 (Invitrogen) or infected by HSV-1 with the indicated titer for 24 h. The supernatant was collected and 50 μL QUANTI-Luc Luciferase reagent (Invivogen) was added to 20 μL supernatants per well, and luciferase luminescence was detected by a microplate reader (BioTek Synergy H1) immediately.
9
Cell Culture and Antibody Reagents
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MDA-MB-231 cells were maintained in the media of DMEM/F12 with 10% FBS, and SUM159 cells were cultured in DMEM/F12 with 10% FBS containing hydrocoretisone (1 μg/ml). The following antibodies were purchased: cGAS (Arigo Biolaboratories); AMPKα, pT172-AMPKα, and pS172-TBK1 (Cell Signaling Technology); TBK1 (Novus Biologicals); β-actin (Santa Cruz Biotechnology), mouse and rabbit secondary antibody-conjugated horseradish peroxidase (Jackson ImmunoResearch); and secondary antibodies for immunofluorescence staining (Invitrogen). RU.521 was purchased from In vivoGen; propidium Iodide, 2’,3’-cGAMP, pan caspase inhibitor (Z-VAD-FMK) and autophagey inhibitor (3-MA and chloroquine) were purchased from Sigma Aldrich; inhibitors of necrosis and ferroptosis (necrostatin-1 and ferrostatin-1) were purchased from Cayman Chemicals. The shRNA clones of ATG5 (TRCN0000330394), cGAS (TRCN0000149984), and STING (TRCN0000134594) were obtained from the RNA Technology Platform and Gene Manipulation Core of the Academia Sinica in Taiwan.
10
Reconstitution and Preparation of Immune Reagents
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