Interleukin 7 (il 7)

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IL-7 is a recombinant protein produced by Thermo Fisher Scientific. It is a cytokine that plays a crucial role in the development and maintenance of T cells and B cells.

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487 protocols using interleukin 7 (il 7)

Differentiation of ILC2 Progenitors

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The ILC2 differentiation potential of hematopoietic progenitors was performed as previously reported (Wong et al., 2012 (link)). In brief, freshly sorted CLPs from the Bcl11b^flox/floxRosa26^{CreERT2/CreERT2} and control Bcl11b^+/+ Rosa26^{CreERT2/CreERT2} mice that were treated with Tam 4 d earlier (4.0 mg Tam by intraperitoneal injection over three consecutive days) were cultured on OP9-DL1 monolayers in the presence of 10 ng/ml IL-7 (PeproTech) and 10 ng/ml IL-33 (PeproTech) for 22 d.
For the short-term fate assay, purified Bcl11b⁻ChILPs and Bcl11b⁺ChILPs were cultured on OP9 monolayers in the presence of 25 ng/ml IL-7 (PeproTech) and 25 ng/ml Stem Cell Factor (SCF; PeproTech) for 6 d as previously described (Constantinides et al., 2014 (link)).
For deleting Bcl11b in ILC2s, sorted ILC2Ps from the BM of Bcl11b^floxfloxRosa26^{CreERT2/CreERT2} and control mice were cultured on OP9-DL1 monolayers in the presence of 20 ng/ml IL-7, 20 ng/ml SCF, and 10 ng/ml IL-2 or 20 ng/ml IL-7 plus 20 ng/ml IL-25 or plus 20 ng/ml IL-33. After 3–5 d, Tam was added in the medium to induce Bcl11b deletion in vitro. Cells were collected and analyzed 10–14 d after Tam treatment.

Yu Y., Wang C., Clare S., Wang J., Lee S.C., Brandt C., Burke S., Lu L., He D., Jenkins N.A., Copeland N.G., Dougan G, & Liu P. (2015). The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development. The Journal of Experimental Medicine, 212(6), 865-874.

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Culturing Intestinal ILC1s and ILC3s

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Freshly sorted small intestinal ILC1s (defined as EYFP⁺/NKp46⁺/CD45^high/CD90^intermediate) or NKp46⁺ ILC3s (defined as EYFP⁺/NKp46⁺/CD45^intermediate/CD90^high) were cultured in IMDM Glutamax supplemented with 10% (vol/vol) FBS, β-ME (55 µM), and penicillin-streptomycin. ILC1s were grown in the presence of (1) IL-7 (5 ng/ml; Peprotech) or (2) mixed IL-7 (5 ng/ml), IL-1β (20 ng/ml), and IL-23 (20 ng/ml). ILC3s were grown in the presence of (1) IL-7 (5 ng/ml; Peprotech) or (2) mixed IL-7 (5 ng/ml) and IL-12 (20 ng/ml). After 20 h, cells were stained as in Flow cytometry and cell sorting.

Krzywinska E., Sobecki M., Nagarajan S., Zacharjasz J., Tambuwala M.M., Pelletier A., Cummins E., Gotthardt D., Fandrey J., Kerdiles Y.M., Peyssonnaux C., Taylor C.T., Sexl V, & Stockmann C. (2022). The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut. The Journal of Experimental Medicine, 219(2), e20210909.

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Generating Memory-like TH17 Cells

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Naive (CD4⁺CD25⁻CD62L^highCD44^low) and memory-like (CD4⁺CD25⁻CD62L^lowCD44^high) T cells from the spleens and lymph nodes of 7–12-week-old mice were isolated using a FACS Aria cell sorter II (BD Biosciences, Franklin Lakes, NJ, USA). Purified naive and memory-like CD4⁺ T cells were stimulated with IL-1β (20 ng/mL, R&D Systems, Minneapolis, MN, USA), IL-23 (20 ng/mL, R&D Systems), IL-7 (10 ng/mL, Peprotech, Rocky Hill, NJ, USA), or plate-bound anti-CD3/CD28 (2 μg/mL, BD Biosciences). Cytokines and other reagents used in further cell culture experiments included TNF (20 ng/mL, Peprotech), IL-12 (10 ng/mL, Peprotech), IL-1RA (100 ng/mL, R&D Systems), Bay 11-7082 (1 μM, Calbiochem, San Diego, CA, USA), SB203580 (1 μM, Calbiochem), and CsA (50 ng/mL, Calbiochem). To generate OT-II memory-like T_H17 cells in vitro, naive (CD4⁺Vα11⁺CD25⁻CD62L^highCD44^low) T cells were sorted from either OT-II or IL-1R1-deficient (Il1r1^−/−) OT-II mice. These CD4⁺ T cells were primed with anti-CD3/CD28 (4 μg/mL) in the presence of TGF-β (0.5 ng/mL, R&D Systems), IL-6 (30 ng/mL, BD Biosciences), and IL-23 (20 ng/mL, BD Biosciences). After a 4-day priming period, the OT-II T_H17 cells were washed and further cultured in medium containing IL-7 (10 ng/mL, Peprotech) for 8–10 days.

Lee H.G., Lee J.U., Kim D.H., Lim S., Kang I, & Choi J.M. (2019). Pathogenic function of bystander-activated memory-like CD4+ T cells in autoimmune encephalomyelitis. Nature Communications, 10, 709.

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OP9 Co-Culture for T Cell Differentiation

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OP9-DL1/OP9-GFP co-culture assays were essentially performed as described previously (48 (link)). Sorted precursors (DN2a and DN2b) were plated onto subconfluent OP9 BM stromal cells expressing either the Notch ligand Delta-like ligand 1 (OP9-DL1) or GFP (OP9-GFP) as a control in a 24-well plate. Cocultures were performed in the presence of 10 ng/mL SCF, 5 ng/mL Flt3-L, and 1 ng/mL IL-7 for OP9-DL1 T cell differentiation and 5 ng/mL IL-7 for OP9-GFP cultures (all obtained from Peprotech). After 4 days, half of the culture medium was replaced with fresh medium and at day 7 of differentiation thymocytes were harvested and separated from contaminating OP9 cells by filtering the cocultured cells through a 50 μm filter (Sysmex) prior to seed onto fresh OP9 monolayers. Lineage differentiation was monitored for up to 15 days. Discrimination of dead cells were performed using 7-amino-actinomycin D according to the manufacturer's instructions.

Kunze-Schumacher H., Winter S.J., Imelmann E, & Krueger A. (2018). miRNA miR-21 Is Largely Dispensable for Intrathymic T-Cell Development. Frontiers in Immunology, 9, 2497.

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Transdifferentiation and Reprogramming of Pre-B Cells

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For transdifferentiation pre-B cells were infected with C/EBPαER-hCD4 retrovirus produced by the PlatE retroviral packaging cell line (Cell Biolabs, # RV-101). The cells were expanded for 48 hr on Mitomycin C-inactivated S17 feeders grown in RPMI medium supplemented with 10 ng/mL each of IL-7 (Peprotech) and hCD4⁺ were sorted (FACSaria, BD). For transdifferentiation C/EBPa was induced by treating the cells with 100 nM β-Estradiol (E2) in medium supplemented with 10 ng/mL each of IL-7, IL-3 (Peprotech) and human colony-stimulating factor 1 (hCSF-1, kind gift of E. Richard Stanley). For reprogramming hCD4⁺ cells were plated at 500 cells/cm² in gelatinised plates (12 wells) on irradiated MEF feeders in RPMI medium and pre-treated for 18 hr with E2 to induce C/EBPα. After E2 washout the cultures were switched to serum-free N2B27 medium supplemented with 10 ng/ml IL-4, IL-7 and IL-15 (Peprotech) at 2 ng/ml and treated with 2 μg/ml of doxycycline to activate OSKM. From day two onwards the N2B27medium was supplemented with 20% KSR (Life Technologies), 3 µM CHIR99021 and 1 µM PD0325901 (2i medium). A step-by-step protocol describing the reprogramming procedure can be found at Nature Protocol Exchange (https://www.nature.com/protocolexchange/protocols/4567).

Francesconi M., Di Stefano B., Berenguer C., de Andrés-Aguayo L., Plana-Carmona M., Mendez-Lago M., Guillaumet-Adkins A., Rodriguez-Esteban G., Gut M., Gut I.G., Heyn H., Lehner B, & Graf T. (2019). Single cell RNA-seq identifies the origins of heterogeneity in efficient cell transdifferentiation and reprogramming. eLife, 8, e41627.

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Western Blot Analysis of Signaling Pathways

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Cells were lysed in RIPA buffer containing phosphatase and protease inhibitor cocktails (Roche). Lysates were resolved by SDS-PAGE followed by Western blot using HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Thermo Scientific). Signal was detected using a Chemidoc MP System and quantified using Image Lab software (Bio-Rad Laboratories); there were no saturated pixels in any quantified images. For IL7 stimulation, CD4⁺ T cells were isolated from LN and stimulated ex vivo with IL7 (Peprotech) for 5 minutes at 37°C. For S1P stimulation, CD4 T cells were isolated from LN and incubated ex vivo with 1 μM S1P (Sigma) for 3 hours at 37°C. The cytoplasmic fraction was isolated using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific), according to the manufacturer’s instructions.

Mendoza A., Fang V., Chen C., Serasinghe M., Verma A., Muller J., Chaluvadi V.S., Dustin M.L., Hla T., Elemento O., Chipuk J.E, & Schwab S.R. (2017). Lymphatic endothelial S1P promotes naïve T cell mitochondrial function and survival. Nature, 546(7656), 158-161.

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Homeostatic Expansion of Naive T Cells

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Naive T cells (CD4⁺ CD44⁻) purified from WT (CD45.1) and AMPK^KO-T (CD45.2) mice were mixed at a 1:1 ratio, CFSE-labelled and seeded in a 96-well plate (10⁵ cell/well). T cells were cultured at 37 °C in RPMI supplemented with 5% heat-inactivated FBS (Sigma-Aldrich), 1% non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 2 mM l-glutamin (Invitrogen), 500 U/ml penicillin/500 μg/ml streptomycin (Invitrogen), and 50 μM β-mercaptoethanol (Sigma-Aldrich). The homeostatic stimuli were provided by 2 × 10⁴ DC purified from autologous C57BL/6 (H-2^b) mice and IL-7 (20 ng/ml, Peprotech). Each 4–5 days of culture, half of the medium volume was refreshed with new IL-7-supplemented medium and freshly purified DC. Cells were analyzed on day 13–15. For antioxidant supplementation, MitoTEMPO (20 µM, Sigma) was added to culture medium and refreshed at the same time than IL-7.
Memory-like Th1 from WT (CD45.1) and AMPK^KO-T (CD45.2) mice were mixed at ratio 1.1 and were cultured with 2 × 10⁴ syngeneic DC and IL-7 (20 ng/ml, Peprotech) for 9 additional days. Half of the medium volume was refreshed with new IL-7-supplemented medium and freshly purified DC at day 5.

Lepez A., Pirnay T., Denanglaire S., Perez-Morga D., Vermeersch M., Leo O, & Andris F. (2020). Long-term T cell fitness and proliferation is driven by AMPK-dependent regulation of reactive oxygen species. Scientific Reports, 10, 21673.

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Protocol for Generating Tumor-Associated Antigen-Specific Cytotoxic T Cells

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TAA-CTLs were generated by stimulation of PBMC with peptide-pulsed DCs at an effector-to-target ratio of 10:1 in SFM (Gibco). The TAA-CTLs were manufactured under GMP conditions by the First Affiliated Hospital of USTC with standard operating procedures and predefined batch release criteria. For initial stimulation, a cytokine mix containing IL-7 (10 ng/mL), IL-12 (10 ng/mL), IL-15 (5 ng/mL), and IL-6 (100 ng/mL, all Peprotech, USA) was added at day 0. Medium was changed and continued to be cultured for 4–6 days if the cell number is less than 3×10⁶/mL, otherwise cells were transfered to new culture well and cultured for another 4–6 days. At days 11–13, the cells were counted and then the loaded DCs, cytokines IL-7, IL-15 (all Peprotech, USA), and IL-2 (50 U/mL, Beijing Sihuan, Beijing, China), were added for the second stimulation, and cultured for 3–4 days continuously to amplify TAA-specific CTL. Contamination of fungi, bacteria, and endotoxin in all the cultured samples were detected during the course of cell culture. At the 21th–25th days, partial cells were frozen and the phenotype was tested and remaining was centrifuged. After removal of the supernatants, the cells were suspended in 100 mL of physiological saline containing 1% albumin and IL-2, then intravenously injected to the patient.

Xue L., Hu Y., Wang J., Liu X, & Wang X. (2019). T cells targeting multiple tumor-associated antigens as a postremission treatment to prevent or delay relapse in acute myeloid leukemia. Cancer Management and Research, 11, 6467-6476.

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OP9-Supported B-ALL Transduction

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OP9 stromal feeder cells were cultured in IMDM supplemented with 10% FBS, 100 µg/ml streptomycin, 100 U/ml penicillin, 100 µM l-glutamine, and 50 µM β-mercaptoethanol and grown at 37°C in 10% CO₂. B-ALL tumor cells were cultured on an OP9 layer in media conditioned with 1ng/µl IL-7 (PeproTech). For tet-on–competent B-ALL transduction, 5 × 10⁶ freshly harvested secondary leukemic spleen cells were spin-infected at 1,200 rpm at 22°C onto TRMPV-shRNA retrovirus-coated plates for 2 h. After centrifugation, cells were incubated in medium for 6 h at 37°C in 10% CO₂ and plated onto OP9 stroma layer supplemented with Doxycycline and IL-7 (PeproTech). Sorted cells were cultured on OP9 in the presence of Doxycycline and analyzed by flow cytometry using BD Fortessa. Cell lysates were Western blotted with anti-Ikaros antibody E-20 (sc-9861; Santa Cruz Biotechnology, Inc.), anti–P120-catenin antibody 98/pp120 (BD), and anti-actin antibody I-19 (sc-1616; Santa Cruz Biotechnology, Inc.).

Witkowski M.T., Hu Y., Roberts K.G., Boer J.M., McKenzie M.D., Liu G.J., Le Grice O.D., Tremblay C.S., Ghisi M., Willson T.A., Horstmann M.A., Aifantis I., Cimmino L., Frietze S., den Boer M.L., Mullighan C.G., Smyth G.K, & Dickins R.A. (2017). Conserved IKAROS-regulated genes associated with B-progenitor acute lymphoblastic leukemia outcome. The Journal of Experimental Medicine, 214(3), 773-791.

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Western Blot Analysis of Signaling Pathways

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