Enriched blood DCs or sorted cord blood cDCs were resuspended at 1×106 cells/mL, PBMCs or tumor digests were resuspended at 5–10×106 cells/mL in DC medium (RPMI GlutaMAX, Gibco, #72 400–021, 1% human serum, Sigma, #H4522, 1% Pen/Strep). 1×105 cells (enriched blood DCs or sorted cord blood cDCs) or 0.5–1×106 cells (PBMCs or tumor digests) in 200 µL/well were treated with the indicated stimuli in a 96-well plate at 37°C in a CO2 incubator. For DC activation in the presence of tumor-conditioned medium, 1:1 diluted supernatant from a 6-hour culture of patient-derived tumor digest in DC medium or a 2-day culture of COR-L105 tumor cell line (ECACC, #92031918) in RPMI GlutaMAX, 10% FBS, 1% Pen/Strep was added to sorted cord blood cDCs during stimulation. The following concentrations were used: 10'000 U/mL huIFN-α (R&D, #11100–1), 10'000 U/mL huIFN-β (R&D, #8499-IF-010), 1 µg/mL huIFN-λ (R&D, #1598-IL-025), 50'000 U/mL huIFN-γ (PeproTech, #300–02), 1 µg/mL Pam3CSK4 (Invivogen, #vac-pms), 10 µg/mL Poly(I:C) (Invivogen, #vac-pic), 0.1 µg/mL LPS (Sigma, #L2880), 1 µM TL8-506 (Invivogen, #tlrl-tl8506), 10 µM CL075 (Invivogen, #tlrl-c75), 10 µM R848 (Invivogen, #vac-r848), 10 µg/mL ssRNA40 (Invivogen, #tlrl-lrna40), 10 µg/mL 2’3’-cGAM(PS)2(Rp/Sp) (Invivogen, #tlrl-nacga2srs).
2 3 cgam ps 2 rp sp
Manufactured by InvivoGen
2'3'-cGAM(PS)2 (Rp/Sp) is a synthetic molecule that consists of a cyclic guanosine monophosphate (cGMP) core with two phosphorothioate-modified guanosine monophosphate (GMP) moieties. It is available in Rp and Sp stereoisomeric forms.
Automatically generated - may contain errors
Lab products found in correlation
Fecl2, by Merck Group (2 mentions)
Cucl2, by Merck Group (2 mentions)
Diabzi, by MedChemExpress (2 mentions)
Ifn β elisa assay, by R&D Systems (2 mentions)
Al2 so4 3, by Merck Group (2 mentions)
Adu s100, by MedChemExpress (2 mentions)
Cgamp, by InvivoGen (2 mentions)
Fecl3, by Merck Group (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Zncl2, by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Cacl2, by Merck Group (2 mentions)
Mncl2, by Merck Group (2 mentions)
Rpmi glutamax, by Thermo Fisher Scientific (1 mentions)
Tl8 506, by InvivoGen (1 mentions)
Cl075, by InvivoGen (1 mentions)
Ssrna40, by InvivoGen (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Huifn α, by R&D Systems (1 mentions)
Tlrl lrna40, by InvivoGen (1 mentions)
5 protocols using 2 3 cgam ps 2 rp sp
1
Stimulation of Dendritic Cells with Diverse Immune Modulators
2
Modulating STING-Mediated Interferon Response
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Mouse bone marrow-derived dendritic cells (BMDCs) were isolated and cultured as reported previously57 (link). Human monocyte cell line THP1 cells expressing hSTINGHAQ were purchased from ATCC (Manassas, VA) and cultured according to ATCC’s instruction. THP1 cells expressing hSTINGR232 (WT), hSTINGH232 (REF) were purchased from Invivogen and cultured according to Invivogen’s instruction. To screen for metal ions for modulating IFN-I response of STING agonists, we seeded 0.1 million BMDCs or THP1 cells per well in 96-well plate, and metal ions (e.g., ZnCl2, KCl, MgCl2, MnCl2, CaCl2, Al2(SO4)3, CuCl2, FeCl2, FeCl3, and CoCl2) (Sigma-Aldrich) at various concentrations ranging 0–500 μM were added with or without 5 μM cGAMP (Invivogen). After 24 h incubation at 37 °C, 5% CO2, the supernatants were collected for IFN-β ELISA assay (R&D). To evaluate the effect of MnCl2 on IFN-I response of STING agonists in various human STING variants, the indicated concentrations of MnCl2 and STING agonists, including cGAMP, CDA (Invivogen), 2’3’-cGAM(PS)2 (Rp/Sp) (Invivogen), ADU-S100 (MedChemExpress) and diABZI (MedChemExpress), were added to 0.1 million THP1 reporter cells in 96-well plate. After 24 h incubation at 37 °C, 5% CO2, the supernatants were collected and assessed for IFN-β by ELISA.
3
Modulating STING-Mediated Interferon Response
4
THP1 Cells Respond to Cyclic Dinucleotides
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Example 4
Experimental Design
THP1 cells were treated with dose response curves of either a cyclic dinucleotide (CDN) alone or in combination with HeLa-PBS conditioned media. The CDNs used were all purchased from Invivogen: 2′3′-cGAMP (tlrl-nacga23), 3′3′-cGAMP (tlrl-nacga), 2′3′-cyclic-di-GMP (tlrl-nacdg23), 3′3′cGAMP-fluorinated (tlrl-nacgaf), c-di-GMP-fluorinated (tlrl-nacdgf), and 2′3′-cGAM(PS)2(Rp/Sp) (tlrl-nacga2srs).
Materials and Methods
THP1-Dual cells (acquired from Invivogen) were seeded at 35,000 cells/well in a 96-well plate (100 μl/well). In a separate treatment plate, a 3-fold dose-response curve of each CDN was prepared, starting at 40 μg/ml, in either plain PBS or in combination with HeLa-PBS conditioned media (conditioned media prepared as previously described). 100 μl of compound treatment was transferred to THP1 cells (1:1 dilution) and cells were incubated for 24 hours. THP1 supernatants were subsequently removed and assayed for IRF activity.
Conclusion
As shown in
5
Culturing HeLa and Human Cancer Cell Lines
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Cells were maintained in DMEM high glucose with GlutaMAX and pyruvate (Gibco, cat. 10569010) with 10% FBS and 1x antibiotic-antimycotic (100 units/mL penicillin, 100 ug/mL streptomycin, 0.25 ug/mL Amphotericin B; Gibco, cat. 15240096) in a humidified incubator at 37°C and 5% CO2. Two strains of HeLa cervical adenocarcinoma cells were used: one line was a kind gift from Dr. Anthony Furano at the National Institute of Diabetes and Digestive and Kidney Diseases, originally gifted from the late Dr. Haig Kazazian and known in the LINE-1 field as HeLa-JMV; the other strain was purchased from ATCC and designated herein as "CCL-2 HeLa ATCC". Additional human epithelial cancer cell lines purchased from ATCC include MCF7 (mammary gland adenocarcinoma), JEG-3 (placental choriocarcinoma), and HCT 116 (colorectal carcinoma). THP-1 human leukemia monocytes were also purchased from ATCC. The STING agonist 2'3'-cGAM(PS)2(Rp/Sp) (Invivogen, cat. tlrl-nacga2srs) and IRF3 inhibitor MRT67307 (Invivogen, cat. inh-mrt) were each diluted with the companion vial of sterile, endotoxin-free LAL water and used at concentrations indicated in figure legends. STING agonists, immunostimulatory dsDNA (pcDNA3.1(+)puro) and CpG-free dsDNA (pCpGfree-mcs, Invivogen) were transfected using Opti-MEM (Gibco, cat.
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