Il 21

Manufactured by Thermo Fisher Scientific

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IL-21 is a recombinant human cytokine that is a member of the common gamma chain receptor cytokine family. It functions as a regulator of T and B cell proliferation and differentiation.

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Human IL-21 Anti-IL-21 e660 Mouse IL-21 ELISA kit Recombinant IL-21 Anti-IL-21 Human IL-21 platinum ELISA kit Mouse recombinant IL-21 IL-21(210–21, Human ELISA Kit IL-18 and IL-21 IFN-γ or IL-21

245 protocols using il 21

Assessing IL-21 Regulation of B Cell Signaling

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Splenic CD19⁺ B cells from BALB/c mice were purified using magnetic mouse CD19 MicroBeads (Miltenyi Biotec) and cultured in flat-bottom 96-well plates (Corning). To assess effects of IL-21 on the expression of c-Myc, BATF, IRF4, p-S6, cyclin D3, and Foxo1, cells were incubated with the indicated concentration of IL-21 (PeproTech) and 20 μg/ml of anti-mouse CD40 (FGK4.5) for 24 h at 37°C. To inhibit Foxo1, cells were treated with AS1842856 (2 μM; MedChem Express) for 1 h, followed by further incubation with 20 μg/ml of anti-CD40 mAb with or without 80 ng/ml of IL-21 for 16 h. To study Foxo1 intracellular localization, cells were rested for 30 min at 37°C, and where indicated, further incubated with 10 μg/ml of goat anti-mouse IgM (μ chain specific; Thermo Fisher Scientific) for 10 min followed by a 30-min stimulation with 80 ng/ml of IL-21.

Petersone L., Wang C.J., Edner N.M., Fabri A., Nikou S.A., Hinze C., Ross E.M., Ntavli E., Elfaki Y., Heuts F., Ovcinnikovs V., Rueda Gonzalez A., Houghton L.P., Li H.M., Zhang Y., Toellner K.M, & Walker L.S. (2023). IL-21 shapes germinal center polarization via light zone B cell selection and cyclin D3 upregulation. The Journal of Experimental Medicine, 220(10), e20221653.

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Isolation and Culture of B Cells

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Magnetic separation (Miltenyi Biotec) was used to purify CD19⁺ B cells from BALB/c or p110δ^D910A spleen. Cells (1 × 10⁶) were cultured for 16 h alone or in the presence of 200 ng/ml IL-21 (PeproTech) or 10 ng/ml IL-4 (PeproTech). For STAT3 inhibition experiments cultures were supplemented with 10, 50, or 100 μM S3I-201 (Calbiochem) as indicated. For PI3K inhibition experiments cultures were supplemented with 10 μM LY-294002 (Invitrogen) as indicated. For assessment of activated STAT proteins cells were cultured for 2 h alone or in the presence of 200 ng/ml IL-21 (PeproTech). For experiments to determine the target of IL-21, 2.5 × 10⁴ BALB/c or IL-21R^−/− B cells were cultured with 2.5 × 10⁴ magnetically separated (Miltenyi Biotec) CD4⁺CD25⁻ T cells from BALB/c or IL-21R^−/− lymph node for 16 h alone or in the presence of 200 ng/ml IL-21 (PeproTech).

Attridge K., Kenefeck R., Wardzinski L., Qureshi O.S., Wang C.J., Manzotti C., Okkenhaug K, & Walker L.S. (2014). IL-21 Promotes CD4 T Cell Responses by Phosphatidylinositol 3-Kinase–Dependent Upregulation of CD86 on B Cells. The Journal of Immunology Author Choice, 192(5), 2195-2201.

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Feeder-Supported In Vitro GC B Cell Culture

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NIH-3T3 feeder cells that stably express CD40L and BAFF were seeded into 96-well plates at 15,000 cells per well in 100 μL complete RPMI-1640 one day before receiving B cells. On day 0, single FAS⁺GL7⁺ GC B cell was sorted directly into these feeder wells, supplemented with recombinant mouse IL-4 (2 ng/mL, Peprotech) and IL-21 (2.5 ng/mL, Peprotech). On day 2, 50 μL culture medium was removed and each well was replenished with 100 μL fresh culture medium containing IL-21 (2.5 ng/mL, Peprotech). From day 3 to 8, 100 μL old medium was replaced with 100 μL fresh medium containing IL-21 (2.5 ng/mL, Peprotech) for each well every day. On day 9 and 10, culture supernatants were harvested (antibody-secreting cell supernatants) and used as cytometry staining or ELISA detection reagent for subsequent assays. These supernatants typically contained ~10 μg/mL total mouse IgG by ELISA. Single-cell cultures in plates were frozen stored at –80 °C until subsequent use for cloning of Ig-coding sequences using a published protocol.^{58 (link)}

Lin Y., Wan Z., Liu B., Yao J., Li T., Yang F., Sui J., Zhao Y., Liu W., Zhou X., Wang J, & Qi H. (2024). B cell-reactive triad of B cells, follicular helper and regulatory T cells at homeostasis. Cell Research, 34(4), 295-308.

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Culturing Cells with IL-21 and CD40L

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IL-21: As described in our earlier publication, IL-21 (100 ng/ml, PeproTech EC, UK) was added to cultures containing 0.16×10⁶ cells/ml.[31] (link) The cultures were readjusted on third day to 0.16×10⁶ cells/ml and IL-21 was re-added. The cells were harvested on the 6^th day for analysis.
CD40 ligand, CD40L: 0.5×10⁶ irradiated (15,000RAD) L or CD40L-L cells were plated in wells of a 24 well plate and used 24 hours later. Equal number of MEC1 and MEC2 cells were seeded on the monolayer and incubated for 3 days at 37°C and 5% CO₂[33] (link).

Rasul E., Salamon D., Nagy N., Leveau B., Banati F., Szenthe K., Koroknai A., Minarovits J., Klein G, & Klein E. (2014). The MEC1 and MEC2 Lines Represent Two CLL Subclones in Different Stages of Progression towards Prolymphocytic Leukemia. PLoS ONE, 9(8), e106008.

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Magnetic Separation of B Cells for IL-21 Signaling

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Magnetic separation (Miltenyi Biotec) was used to purify CD19+ B cells from BALB/c or p110δ^D910A spleen. 1×10⁶ cells were cultured for 16 hours alone or in the presence of 200ng/ml IL-21 (Peprotech) or 10ng/ml IL-4 (Peprotech). For STAT3 inhibition experiments cultures were supplemented with 10, 50 or 100μM S3I-201 (Calbiochem) as indicated. For PI3K inhibition experiments cultures were supplemented with 10μM LY-294002 (Invitrogen) as indicated. For assessment of activated STAT proteins cells were cultured for 2 hours alone or in the presence of 200ng/ml IL-21 (Peprotech). For experiments to determine the target of IL-21 2.5×10⁴ BALB/c or IL-21R−/− B cells were cultured with 2.5×10⁴ magnetically separated (Miltenyi Biotec) CD4+CD25− T cells from BALB/c or IL-21R−/− lymph node for 16 hours alone or in the presence of 200ng/ml IL-21 (Peprotech).

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Cytokine Stimulation of Splenocytes

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Spleens were aseptically removed from naïve WT, Il21r^-/-, STAT3^flox/flox or CD4^stat3-/- mice and cells were mechanically disrupted through a 100 μm cell strainer (BD Biosciences, San Jose, CA) using the plunger of a 6 ml syringe. RBCs were lysed in ACK lysing buffer (Lonza, Walkersville, MD), and the remaining cells were washed twice with ice-cold PBS. A total of 5×10⁶ splenocytes were cultured in duplicate in 1 ml of RPMI 1640 (Gibco) supplemented with 10% FBS and 100 μg/ml penicillin/streptomycin (all from Gibco). Cells were allowed to rest for 2 additional hours at 37°C and subsequently were stimulated in the presence of recombinant murine IFN-γ, IL-17A, IL-21 and IL-22 (20 ng/ml; Peprotech, Rocky Hill, New Jersey, USA) alone or in combination (IFN-γ+IL-17A; IFN-γ+IL-21; IFN-γ+IL-22; 20 ng/ml each). Cells receiving no cytokine treatments were used as controls. Cells were harvested at different intervals following the addition of cytokines and were stained with indicated antibodies.

Solaymani-Mohammadi S, & Berzofsky J.A. (2019). Interleukin 21 collaborates with interferon-γ for the optimal expression of interferon-stimulated genes and enhances protection against enteric microbial infection. PLoS Pathogens, 15(2), e1007614.

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Investigating CD4+ T Cell Response in IL-21R-/- Mice

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CD19⁺ B cells from BALB/c or IL-21R^−/− spleen were isolated by magnetic separation (Miltenyi Biotec) and cultured for 16 h with 1 μg/ml OVA peptide. Peptide-pulsed cells (3 × 10⁶) were injected i.v. into IL-21R^−/− recipients. magnetic separation (Miltenyi Biotec) was used to purify CD4⁺ cells from IL-21R^−/− DO11.10 TCR⁺ lymph node. Twenty-four hours after B cell transfer, a total of 2 × 10⁶ CellTrace Violet–labeled IL-21R^−/− DO11.10 TCR⁺ T cells were injected i.v. and 1 μg IL-21 (PeproTech) or PBS i.p. Where indicated recipients were also given 100 μg anti-CD86 (Bio X Cell) or PBS i.p. immediately after T cell transfer. Mice were culled at day 7 for analysis of inguinal lymph node and splenic cells.

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Expansion and Transduction of CD8+ T Cells

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CD8⁺CD161⁺ cells, CD8⁺CD161⁻ cells, and bulk PBMC isolated from normal donor apheresis products were stimulated with plate bound anti-CD3/CD28 (eBioscience) and recombinant Clec2d (R&D Systems) and expanded in RPMI-1640, 10% fetal bovine serum, and 2 mmol/L GlutaMAX (Invitrogen) in a cytokine cocktail comprised of 10 ng/ml IL-7, 5 ng/ml IL-15, and 30 ng/ml IL-21 (all from Peprotech), given that IL-21 was important for maintenance of CD161 expression and phenotype. In some experiments, a second bulk PBMC control was expanded using the standard expansion protocol of only 10 ng/ml IL-7 and 5 ng/ml IL-15 to demonstrate that the presence of IL-21 during expansion made no functional impact at all upon the function of CAR-transduced bulk PBMC. After 48 hours, T cell blasts were harvested and added to retronectin (Takara Bio USA) coated plates onto which retroviral supernatants had previously been affixed by centrifugation at 2,000 × G for 60 minutes. After an additional 48 hours, transduced blasts were moved to tissues culture plates and expanded an additional 10 to 15 days in the IL-7/15/21 cocktail.

Konduri V., Joseph S.K., Byrd T.T., Nawas Z., Vazquez-Perez J., Hofferek C.J., Halpert M.M., Liu D., Liang Z., Baig Y., Salsman V.S., Oyewole-Said D., Tsimelzon A., Burns B.A., Chen C., Levitt J.M., Yao Q., Ahmed N.M., Hegde M, & Decker W.K. (2021). A Subset of Cytotoxic Effector Memory T cells Enhances CAR T cell Efficacy in a Model of Pancreatic Ductal Adenocarcinoma. Science translational medicine, 13(592), eabc3196.

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Cytokine-induced CD4+ T cell activation

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Naïve CD4⁺ T cells (1 ×10⁵ cell/well) from HC subjects (n = 6) were stimulated with dynabeads^® human T-activator CD3/CD28 (Invitrogen, USA) in flat-bottom 96-well plates, and cultured in the presence of IL-12 (PeproTech, USA) (10 ng/ml), IL-21 (PeproTech, USA) (20 ng/ml), IL-12 + IL-21, or IL-17 (PeproTech, USA) (10 ng/ml) for 72 hours, respectively.

Du B., Teng J., Yin R., Tian Y., Jiang T., Du Y, & Cai W. (2021). Increased Circulating T Follicular Helper Cells Induced via IL-12/21 in Patients With Acute on Chronic Hepatitis B Liver Failure. Frontiers in Immunology, 12, 641362.

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B cell differentiation with CD4+ T cells

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Human B cells were stained with 0.25 μM CFSE (Invitrogen) and plated out into a 96-well V-bottom plate (5x10⁴ cells/well) in triplicate. Cells were stimulated for 5 days with 5 μg/mL anti-IgM (Jackson ImmunoResearch), 100 ng/mL anti-CD40 (R&D) and 10 ng/mL IL-21 (PeproTech) final concentration.
For the B cell co-culture with CD4+CD45RO+CXCR5+ T cells, CD4+ T cells were first enriched by positive selection using CD4 microbeads (Miltenyi Biotech), then stained with CD45RO (BD) and CXCR5 (Biolegend) antibodies in PBS-0.5% BSA-2 mM EDTA. CD4+CD45RO+CXCR5+ were FACS sorted on an ARIA-III (BD Bioscience).
CFSE stained B cells and CD4+CD45RO+CXCR5+ T cells were plated out into a 96-well V-bottom plate at a 1:1 ratio and cells were stimulated with SEB 10 ng/mL (SIGMA-Aldrich), IL-21 10 ng/mL and BAFF 10 ng/mL (PeproTech) final concentration. Cells were incubated for 5 days.
Supernatants were collected and antibody production was analysed using a multiplex isotyping Panel 1 kit (Meso Scale Discovery).

Dumont C., Sivars U., Andreasson T., Odqvist L., Mattsson J., DeMicco A., Pardali K., Johansson G., Yrlid L., Cox R.J., Seeliger F., Larsson M., Gehrmann U., Davis A.M, & Vaarala O. (2020). A MALT1 inhibitor suppresses human myeloid DC, effector T-cell and B-cell responses and retains Th1/regulatory T-cell homeostasis. PLoS ONE, 15(9), e0222548.

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