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).
Ssrna40
Manufactured by InvivoGen
Sourced in United States, France
SsRNA40 is a synthetic single-stranded RNA molecule. It functions as a toll-like receptor 7 (TLR7) agonist, which can be used to stimulate immune responses in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pam3csk4, by InvivoGen (10 mentions)
Poly 1 c, by InvivoGen (6 mentions)
Flagellin, by InvivoGen (5 mentions)
Imiquimod, by InvivoGen (5 mentions)
Lipopolysaccharides (lps), by InvivoGen (5 mentions)
Fsl 1, by InvivoGen (4 mentions)
Lsm 510, by Zeiss (4 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Ssrna41, by InvivoGen (3 mentions)
Lyovec, by InvivoGen (3 mentions)
Calcium ionophore a23187, by Merck Group (2 mentions)
Sytox orange nucleic acid stain, by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by BioLegend (2 mentions)
Anti human cd14 magnetic beads, by Miltenyi Biotec (2 mentions)
Gm csf, by BioLegend (2 mentions)
Active tgfβ, by Thermo Fisher Scientific (2 mentions)
Stemxvivo serum free dc base medium, by Bio-Techne (2 mentions)
Anti tgfβ antibody clone 1d11, by BioXCell (2 mentions)
25 protocols using ssrna40
1
Stimulation of Dendritic Cells with Diverse Immune Modulators
2
Visualizing NETosis in Neutrophils
3
Isolation and Stimulation of Splenic B Cells
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Splenic B cells were purified with anti-CD19 mAb–coated microbeads or the Pan B cell isolation kit (Miltenyi Biotec) or by means of cell sorting with the FACSAria Fusion (BD Bioscience). B cells (2 × 106 cells/ml) were resuspended in complete medium [RPMI 1640 media containing 10% fetal bovine serum, penicillin (200 μg/ml), streptomycin (200 U/ml), 4 mM l -glutamine, and 5 × 10−5 M 2-mercaptoethanol (all from Gibco)]. B cells were stimulated with an agonistic anti-CD40 mAb (10 μg/ml; FGK45, Enzo Life Sciences), BAFF (100 ng/ml; R&D Systems), LPS (10 μg/ml; Escherichia coli serotype 0111: B4, Sigma-Aldrich), or other TLR agonists (TLR1, Pam3CSK4, 300 ng/ml; TLR2, heat-killed Listeria monocytogenes, 108 cells/ml; TLR3, polyriboinosinic acid/polyribocytidylic acid, 10 μg/ml; TLR5, Salmonella Typhimurium flagellin, 1 μg/ml; TLR6, Pam2CGDPKHPKSF, 100 ng/ml; TLR7, ssRNA40/LyoVec, 5 μg/ml; and TLR9, ODN1826, 5 μM; Invivogen).
4
Lentiviral Vector Production Protocols
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SIV3+Vpx+, SIV3+Vpx-, and VSV-G plasmids were provided by Andrea Cimarelli (Université de Lyon, Lyon, France). The lentiviral packaging plasmid psPAX2 was obtained from Addgene. LPS was derived from Escherichia coli O55:B5 (Sigma-Aldrich). Pam3CSK4, Poly IC HMW, ssRNA40, and R837 were purchased from InvivoGen.
5
Dose-response curves of immune cell stimulation
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Dose–response and timing curves were performed for each stimulus. Ficoll-separated PBMCs (4 × 106 cells/well) and MDMs (5 × 105 cells/well) were stimulated for 24 h with LPS (50 µg/mL), peptidoglycan (PGN) (10 µg/mL), lipoteichoic acid (LTA) (1 µg/mL), ssRNA40, a uridine-rich ssRNA analog of HIV-1 ssRNA (6,25 µg/mL) (InvivoGen, San Diego, CA, USA), interferon-γ (IFNγ) (100 U/mL), anti-CD28 (1.25 µg/mL) (R&D System, Minneapolis, MN, USA), and anti-CD3 (2.5 µg/mL) (BD Pharmigen, San Diego, CA, USA). After 24 h, the cells were harvested for flow cytometry analyses; the supernatants of a subset of 5 HIV−, 5 HIV+ untreated, and 11 HIV + cART (5 INRs and 6 FRs) patients were collected and stored for the Luminex assay.
6
RNA Transfection in THP-1 Cells
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RNA including negative control tRNA, positive control Poly I:C, HCV 1–807 bp, 2406–3256 bp, 5626–6437 bp, HCV 3′UTR, HCV full length (FL) RNA, ssRNA40, ssRNA41 and ssPolyU (Invivogen, USA) were transfected with Lipofectamine 2000 (Invitrogen, USA) diluted in OptiMEM (Invitrogen, USA) without nucleic acids according to the manufacturer’s protocol. 1 µg of nucleic acid were delivered into THP-1 cells or THP-1 derived macrophages with 2.5 µl of Lipofectamine 2000 unless described otherwise.
7
Monocyte-Derived Dendritic Cell Differentiation
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Leukocyte apheresis cones were collected from healthy donors at the National Blood Service (Manchester, UK). PBMCs were separated by centrifugation using Ficoll-Paque (GE Healthcare, Amersham, UK). Monocytes were separated from PBMCs using anti-human-CD14 magnetic beads (Miltenyi Biotec, Cologne, Germany) according to the manufacturer’s instructions, using an LS MACS separation column. Monocyte purity was consistently over 95%.
Monocytes were cultured in StemXvivo serum-free DC base medium (Bio-techne, Minneapolis, MN, USA) containing 25 ng ml−1 GM-CSF and 25 ng ml−1 interleukin-4 (Biolegend, San Diego, CA) for 6 days at a concentration of 0.5 × 106 cells per ml in 24-well tissue culture-treated plates. Half of the medium was removed on day 3 and replaced with fresh medium and cytokines. After 6 days of differentiation, cells were treated for 48 h with different combinations of compounds: 5 ng ml−1 active TGFβ (Peprotech, Rocky Hill, NJ), 100 μg ml−1 anti-TGFβ antibody (clone 1D11, West Lebanon, BioXcell, NH), 1 µM pan-RA receptor antagonist (LE540, a kind gift from Hiroyuki Kagechika, Tokyo Medical and Dental University, Tokyo, Japan), 100 nM RA (Sigma Aldrich, St Louis, MO, USA), 1 µg ml−1 Pam3CSK4, 10 μg ml−1 Poly(I:C), 1 µg ml−1 flagellin, 5 μg ml−1 Imiquimod, 5 μg ml−1 ssRNA40, or 100 ng ml−1 LPS (all from Invivogen, San Diego, CA).
Monocytes were cultured in StemXvivo serum-free DC base medium (Bio-techne, Minneapolis, MN, USA) containing 25 ng ml−1 GM-CSF and 25 ng ml−1 interleukin-4 (Biolegend, San Diego, CA) for 6 days at a concentration of 0.5 × 106 cells per ml in 24-well tissue culture-treated plates. Half of the medium was removed on day 3 and replaced with fresh medium and cytokines. After 6 days of differentiation, cells were treated for 48 h with different combinations of compounds: 5 ng ml−1 active TGFβ (Peprotech, Rocky Hill, NJ), 100 μg ml−1 anti-TGFβ antibody (clone 1D11, West Lebanon, BioXcell, NH), 1 µ
8
Investigating Immune Cell Responses to TLR Agonists
9
Isolation and Characterization of Murine pDCs
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Bone marrow (BM) cells removed from tibiae, femurs and hips of 8- to 14-week-old C57BL/6J mice were incubated in low endotoxin RPMI-1640 medium (Fisher Scientific, Illkirch, France) supplemented with 10% (vol/vol) FCS and 1% antibiotics (penicillin and streptomycin) for 18 h with 1 μg/ml of the oligodeoxynucleotide CpG 1668 (CpG-B) (Eurogentec, Angers, France) or with the respective agonists of TLR1-9, supplied in a commercial kit (InvivoGen, Toulouse, France), including: Pam3CSK4 (0.5 μg/ml), FSLI (1 μg/ml), HKLM (2 × 106 cells/ml), Poly I:C (HMW) (5 μg/ml), Poly I:C (LMW) (5 μg/ml), LPS-EK standard (1 μg/ml), FLA-ST standard (1 μg/ml), ssRNA40/LyoVec (2 μg/ml) as well as CpG 1826 (CpG-B) (1 μg/ml). c-kit+ BM cells were sorted by immuno-magnetic separation using a RoboSep automaton (StemCell Technologies, Grenoble, France). Sorted cells were further stained with appropriate fluorochrome-conjugated mAbs against Sca-1, B220 (BD Biosciences, Le Pont de Claix, France), PDCA-1 (eBioscience, Paris, France or BioLegend, London, UK) and electronically sorted as a c-kit+Sca-1+B220+PDCA-1+ cell subset using a FACS-Aria IIIu (BD Biosciences).
10
Monocyte cytokine response to TLR ligands
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IHMC monocytes were purified using CD14+ Microbeads (Miltenyi) and cultured for 24h in the presence of TLR ligands (TLR2: 108/ml heat-killed Listeria monocytogenes, TLR3: 10 µg/ml polyI:C, TLR4: 1 µg/ml E. coli LPS, TLR5: 1 µg/ml B. subtilis flagellin, TLR8: 5 µg/ml ssRNA40; Invivogen). Supernatants were collected and measured for the accumulation of 12 different cytokines and chemokines using Cytometric Bead Arrays (BD Biosciences).
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