Ril 7

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The RIL-7 is a laboratory equipment designed for automated liquid handling. It features precise pipetting and dilution capabilities to support various liquid-based applications in scientific research and testing.

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Lab products found in correlation

13 protocols using ril 7

Expansion of HPV-reactive T cells

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PBMCs and TILs from 17 patients with HPV-positive HNSCC were used for in vitro T cell expansion. Potential CD8 T cell epitopes derived from HPV proteins E2, E5, E6 and E7 and presented by a reference set of 27 human leukocyte antigens (HLA-A, B and C) covering 97% of the population were predicted using the Immune Epitope Database (IEDB)^{30 (link)}. A total of 251 predicted 9–10-amino-acid-long peptides with a percentile rank of less than or equal to 1 (Supplementary Table 2) were synthesized as crude material (A&A Labs) and ultimately resuspended in DMSO. A peptide pool was prepared containing all 251 peptides from proteins E2 (125 peptides), E5 (55 peptides), E6 (50 peptides) and E7 (21 peptides). PBMCs were cultured in complete CTS OpTmizer medium (CTS OpTmizer T Cell Expansion SFM with CTS supplement A1048501, substituted with l-glutamine, penicillin–streptomycin and 2% human serum, Sigma-Aldrich, H3667) in the presence of the HPV-peptide pool (1 μg ml⁻¹ per peptide), rIL-2 (Peprotech, 50 IU ml⁻¹), rIL-7 (Peprotech, 25 ng ml⁻¹) and rIL-15 (Peprotech, 25 ng ml⁻¹). HPV-peptides were only added on the first day of culture, whereas cytokines were supplemented whenever cells were split during the two-week expansion period. At day 13 of cell culture, expanded cells were washed and rested overnight in cytokine-free medium.

Eberhardt C.S., Kissick H.T., Patel M.R., Cardenas M.A., Prokhnevska N., Obeng R.C., Nasti T.H., Griffith C.C., Im S.J., Wang X., Shin D.M., Carrington M., Chen Z.G., Sidney J., Sette A., Saba N.F., Wieland A, & Ahmed R. (2021). Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer. Nature, 597(7875), 279-284.

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Mouse iPSCs Differentiation into T Lineage

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Mouse iPSCs were maintained on feeder layers of irradiated SNL76/7 cells in 6-well culture plates (Nunc) and passaged every 3 days. In brief, iPSCs were maintained in DMEM culture medium (0.1 mmol/L nonessential amino acids, 1 mmol/L L-glutamine from Invitrogen (Carlsbad, CA), and 0.1 mmol/L β-mercaptoethanol from Sigma-Aldrich (St. Louis, MO) supplemented with 15% fetal calf serum (FCS) from HyClone, (Logan, UT). Monolayers of OP9-DL1/DL4/I-A^b cells were cultured in α-MEM medium supplemented with 20% FCS and 2.2 g/L sodium bicarbonate from Invitrogen (Carlsbad, CA). Mouse iPSCs were washed once in DMEM culture medium before plating onto sub confluent OP9-DL1/DL4/I-A^b monolayers for T lineage differentiation in the presence of murine rFlt-3 ligand (5 ng/ml) and rIL-7 (1 ng/ml) from Peprotech (Rocky Hill, NJ)15 (link).

Haque M., Song J., Fino K., Sandhu P., Song X., Lei F., Zheng S., Ni B., Fang D, & Song J. (2016). Stem cell-derived tissue-associated regulatory T cells ameliorate the development of autoimmunity. Scientific Reports, 6, 20588.

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Murine Th Cell Differentiation Assay

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Allophycocyanin-conjugated anti-CD4 monoclonal antibody (mAb, GK1.5), anti-IL-4 mAb (11B11), anti-IFN-γ mAb (R4-6A2), anti-CD62L mAb (MEL-14), anti-CD44 mAb (IM7), PE-conjugated anti-IL-4 mAb (BVD4-1D11), PE-conjugated KJ1-26 (anti-clonotypic mAb for DO11.10 TCR, KJ1-26), anti-CD11c mAb (HL3), unconjugated anti-IL-4 mAb (11B11), anti-IL-12 mAb (C17.8), anti-IFN-mAb (R4-6A2), anti-CD44 mAb (IM7), FITC-conjugated anti-CD49b mAb (DX5), and PerCP-conjugated anti-CD4 mAb (GK1.5) were purchased from BD Bioscience. Anti-STAT5 Abs (C-17), anti-STAT6 Abs (N-20), anti-Bcl6 Abs (N-3), anti-tubulin Abs (H-235), and normal rabbit IgG were purchased from Santa Cruz Biotechnology. FITC-conjugated anti-T1/ST2 (IL-33R) mAb (DJ8) was purchased from MD Bioproducts. Mouse rIL-2, rIL-4, rIL-7, rIL-12, and rIL-33 were purchased from PeproTech. Anti-CD3ε mAbs (145-2C11) were purchased from Cedar Lane. Anti-CD28 mAbs (PV-1) were purchased from Southern Biotechnology. The ovalbumin (OVA) peptide (Loh15: residues 323–339; ISQAVHAAHAEINEAGR) was synthesized by BEX Co. Ltd. (Tokyo, Japan). The Bcl6 inhibitory peptide was synthesized by Scrum Inc. (Tokyo, Japan).

Ogasawara T., Kohashi Y., Ikari J., Taniguchi T., Tsuruoka N., Watanabe-Takano H., Fujimura L., Sakamoto A., Hatano M., Hirata H., Fukushima Y., Fukuda T., Kurasawa K., Tatsumi K., Tokuhisa T, & Arima M. (2018). Allergic TH2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4+ T Cells. Frontiers in Immunology, 9, 750.

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EBNA1-specific T cell stimulation

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Immature MoDCs were cultured for 18 h in the presence of recombinant EBNA1 (1μg/ml; Prospec, Rehovot, Israel) and then subjected to maturation, as described above.
CD4⁺ and CD8⁺ T cells (purity >90%) were purified from healthy donors’ PBMCs by immunomagnetic enrichment with CD4 or CD8 microbeads (Miltenyi Biotec). CD4⁺ or CD8⁺ T cells were cultured in 96-well plates (Corning) at a 10:1 cell ratio with autologous EBNA1-pulsed DCs in RPMI 1640 supplemented with 10% heat-inactivated human AB serum and 5 ng/ml rIL-7 (PeproTech, London, U.K.). Lymphocytes underwent three weekly stimulation rounds with autologous EBNA1-pulsed DCs. After the last stimulation, rIL-2 (20 U/ml; Proleukin/Chiron Italia, Milan, Italy) or rIL-15 (10 ng/ml; Immunotools) was added to the culture medium of CD4⁺ and CD8⁺ T cells, respectively. Functional analyses of CD4⁺ and CD8⁺ T cell blasts were performed 7 d after the third round of stimulation.

Morandi F., Di Carlo E., Ferrone S., Petretto A., Pistoia V, & Airoldi I. (2014). IL-27 in Human Secondary Lymphoid Organs Attracts Myeloid Dendritic Cells and Impairs HLA Class I–Restricted Antigen Presentation. Journal of immunology (Baltimore, Md. : 1950), 192(6), 2634-2642.

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Differentiation and Transduction of γδT17 Cells

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Pooled spleen and LN cells were cultured at 1 × 10⁶ cells per ml in RPMI 1640 containing 10% FCS, antibiotics, 1 × Glutamax (Gibco), 10 mM HEPES (SA Pathology), 1 mM sodium pyruvate, 54 pM β-mercaptoethanol and 1 × non-essential amino acids (Gibco) with 5 ng ml⁻¹ recombinant (r)IL-23 (eBioscience), 5 ng ml⁻¹ rIL-1β (Miltenyi Biotec) and 10 μg ml⁻¹ α-IFN-γ (BioXCell) in 96-well round-bottom plates coated with 1 μg ml⁻¹ α-TCR-γδ (clone GL3; Biolegend) for 3 days. Cells were washed and re-seeded on fresh plastic at 1 × 10⁶ cells per ml for a further 3 days as above without TCR-γδ stimulation. Cells were then washed and re-seeded in 20 ng ml⁻¹ rIL-7 (Peprotech) and 10 μg ml⁻¹ α-IFN-γ for a further 3 days. pMIG, pMIG-Rorc and pMIG-Ccr6 (cloned from mouse Ccr6 cDNA) were transfected into EcoPack 2 293 cells (Clontech; mycoplasma free) with Lipofectamine 2000 (ThermoFisher), and supernatant collected after 48 h. γδT17 cells at days 4 and 5 of culture were centrifuged at 2,500 r.p.m. (30 °C for 1.5 h) in supernatant with 8 μg ml⁻¹ polybrene (Sigma) in flat-bottom 96 well trays before being returned to culture.

McKenzie D.R., Kara E.E., Bastow C.R., Tyllis T.S., Fenix K.A., Gregor C.E., Wilson J.J., Babb R., Paton J.C., Kallies A., Nutt S.L., Brüstle A., Mack M., Comerford I, & McColl S.R. (2017). IL-17-producing γδ T cells switch migratory patterns between resting and activated states. Nature Communications, 8, 15632.

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In vitro differentiation of LTi cells

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In vitro LTi cell differentiation was described previously (Cherrier et al., 2012 (link)). Briefly, one day prior to sorting, OP9 cells were plated into 24-well plates at a density of 3.0 × 10⁴ cells per well. Fetal livers were harvested on E13.5-E14.5, and single-cell suspensions were obtained. Negative selection was performed to remove CD3⁺, Ter119⁺, Gr-1⁺, and CD11c⁺ cells, and then CD3⁻CD19⁻B220⁻Gr1⁻CD45⁺cKIT^intCD127⁺α4β7⁺ cells were sorted and ~1000 fetal liver progenitor cells were plated onto OP9 stromal cells along with 10 ng/μl of rSCF (Peprotech catalog no. 250–03) and rIL-7 (Peprotech catalog no. 217–17). A half-media change was performed on days 4 and 11. Cells were passaged onto new OP9 stromal cells on day 7. Analysis by flow cytometry was performed 6 or 14 days after sorting and co-culture.

Bennion B.G., Croft C.A., Ai T.L., Qian W., Menos A.M., Miner C.A., Frémond M.L., Doisne J.M., Andhey P.S., Platt D.J., Bando J.K., Wang E.R., Luksch H., Molina T.J., Roberson E.D., Artyomov M.N., Rösen-Wolff A., Colonna M., Rieux-Laucat F., Di Santo J.P., Neven B, & Miner J.J. (2020). STING Gain-of-Function Disrupts Lymph Node Organogenesis and Innate Lymphoid Cell Development in Mice. Cell reports, 31(11), 107771.

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Expansion of Uterine and Lung γδ17 Cells

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Uterine and lung γδ17 cells were expanded using a protocol adapted from McKenzie et al.^{57 (link)} Uterine or lung cell suspensions were cultured at a 1 × 10⁶ cells/mL in phenol red-free RPMI containing 10% charcoal-treated FCS, glutamine, Pen/Strep, 1 mM pyruvate, β-mercaptoethanol, and 1X NEAA (complete medium), supplemented with 5 ng/mL rIL-23 (eBioscience), 5 ng/mL rIL-1β (R&D Systems), 10 μg/mL anti-IFN-γ (eBioscience), and 1 μg/mL indomethacin (Sigma-Aldrich), for 3 days in 96-well plates coated with 1 μg/mL anti-TCRδ (eBioscience). Cells were harvested and re-plated for 3 days in the same medium, without TCR stimulation. Finally, cells were harvested and incubated for 3 days in complete medium with 20 ng/mL rIL-7 (Peprotech). Throughout the culture protocol, 1 μM progesterone (Sigma-Aldrich) or DMSO were added.

Monin L., Ushakov D.S., Arnesen H., Bah N., Jandke A., Muñoz-Ruiz M., Carvalho J., Joseph S., Almeida B.C., Green M.J., Nye E., Hatano S., Yoshikai Y., Curtis M., Carlsen H., Steinhoff U., Boysen P, & Hayday A. (2020). γδ T cells compose a developmentally regulated intrauterine population and protect against vaginal candidiasis. Mucosal Immunology, 13(6), 969-981.

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Isolation and Characterization of ILC2 Cells

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Lineage-positive lung CD45⁺ cells were eliminated using lineage markers (CD3e, CD4, CD5, CD8a, CD11c, CD19, F4/80, Ly-6G, and NK1.1), and Thy1.2⁺ cells were isolated as ILC2 cells. To assess the levels oILC2-derived cytokines in papain-treated mice, isolated ILC2 cells were cultured in RPMI medium containing 10 ng/mL of recombinant murine IL-7 (rIL-7) (Peprotech, Cranbury, NJ, USA) for 12 h, and cytokine and chemokine levels in the supernatant were measured using a MAGPIX Multiplexing System (Luminex, Austin, TX, USA) and a MLLIPLEX Mouse High Sensitivity T Cell Panel (Merck, Darmstadt, Germany).
To investigate the role of calcium signaling in ILC2 cells, isolated ILC2 cells were stimulated with 10 ng/mL of recombinant murine IL-33 (Biolegend), 30 ng/mL of PMA (Sigma-Aldrich), 500 ng/mL of ionomycin (Sigma-Aldrich), or 10 nM of LTB₄, LTC₄, LTD₄, or LTE₄ (Cayman Chemical) in the presence of 10 ng/mL of rIL-7 (Peprotech) for 40 h. Stimulation was performed with or without FK506 (1000 nM). Cytokine levels were determined as previously described (25 (link)).

Tomiaki C., Miyauchi K., Ki S., Suzuki Y., Suzuki N., Morimoto H., Mukoyama Y, & Kubo M. (2022). Role of FK506-sensitive signals in asthmatic lung inflammation. Frontiers in Immunology, 13, 1014462.

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Autologous DC-T Cell Co-culture

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After 7 days of culture, iDCs, mDCs, mDC_A, mDC_B, and mDC_P were harvested, the supernatants stored, and cells were washed and resuspended in X-VIVO 15 (Gibco, Lonza), and subsequently pulsed with 1 mM MART-1 peptide (ELAGIGILTV), EBV BLMF1 peptide (GLCTLVAML), PDL101 peptide (LLNAFTVTV), or CMV pp65 peptide (NLVPMVATV) (all by KJ Ross Petersen Aps, Copenhagen, Denmark) at 37◦C for 2 h. Hereafter DCs (0.5 × 10⁵) were washed twice and incubated in 48-wells culture plates together with autologous CD8⁺ T cells (0.5 × 10⁶). The general informations and throubleshooting advices were taken from Wolfl et al.^{53 (link)}. For each condition (iDCs, mDCs, mDC_A, mDC_B, mDC_P) were plated 3–5 co-culture replica in order to be able to exclude possible technical errors. At day 3 and 6, half of the medium was refreshed with new containing rIL-2 (50 U/ml, Novartis) and rIL-7 (10 ng/ml, Peprotech, Rocky Hill, NJ). For experiments with supplement of exogenous rIL-12, rIL-6 and rIL-23 (10 ng/ml; Peprotech, Rocky Hill, NJ) were added together with the medium refreshment at day 3 and 6 of the co-culture.

Nastasi C., Fredholm S., Willerslev-Olsen A., Hansen M., Bonefeld C.M., Geisler C., Andersen M.H., Ødum N, & Woetmann A. (2017). Butyrate and propionate inhibit antigen-specific CD8+ T cell activation by suppressing IL-12 production by antigen-presenting cells. Scientific Reports, 7, 14516.

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Retroviral Transduction of BCR-ABL1 into Mouse BM

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A retroviral vector encoding BCR-ABL1^p210-ires-GFP was transfected into the ecotropic packaging cell line PLAT-E and 24 h later supplemented with 10 mM sodium butyrate (Sigma) to augment viral production. The supernatant was collected 24 h later, concentrated using Vivaspin 20 (GE Healthcare) and used to infect BM cells. BM cells were isolated from the long bones of C57BL6/J or Aicda^−/− mice and cultured in StemSpan medium (StemCell technologies) for 48 h with 50 ng/mL Flt3L and 10 ng/mL of rIL-7 (Preprotech). Pelleted BM cells were resuspended in 0.2 mL of concentrated viral supernatant containing 8 μg/mL polybrene (Sigma) and incubated for 2 h before adding 1 mL of StemSpan medium with cytokines, spinning for 30 min at 600 x g and then incubating overnight in 6-well plates. One million cells were then injected in the tail vein of sublethally irradiated (5 Gy) NRG mice. The visible efficiency of infection at the time of injection was typically 5%. Mice were monitored daily and sacrificed when moribund or reaching one of the endpoints (paralysis, rapid loss of weight, ataxia, lack of cleaning, general posture, visible masses). Cells from the BM, blood, spleen, brain and visible masses were isolated using a 70 μM filter and analyzed by flow cytometry or cultured for 48 h in complete IMDM media at 2×10⁶/mL in 6 well plates for RNA isolation.

Montamat-Sicotte D., Liztler L.C., Abreu C., Safavi S., Zahn A., Orthwein A., Muschen M., Oppezzo P., Muñoz D.P, & Di Noia J.M. (2015). HSP90 inhibitors decrease AID levels and activity in mice and in human cells. European journal of immunology, 45(8), 2365-2376.

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