Ril 21

Manufactured by Thermo Fisher Scientific

Sourced in United States

The RIL-21 is a laboratory instrument designed for the extraction and purification of nucleic acids. It utilizes a centrifugation-based process to isolate DNA, RNA, or other genetic material from a variety of sample types. The RIL-21 is capable of handling multiple samples simultaneously and provides consistent, high-quality results.

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Lab products found in correlation

8 protocols using ril 21

C. muridarum Respiratory Infection Model

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Chlamydia muridarum (C. muridarum), obtained from Dr. Xi Yang (the University of Manitoba, Canada), was cultured, purified, and enumerated as previously described [27 (link)]. For C. muridarum respiratory infection animal model, mice were anesthetized via inhalation of isoflurane and then intranasally inoculated with 1 × 10³ inclusion forming units (IFUs) of C. muridarum in 40 μl sucrose-phosphate-glutamic acid (SPG) buffer. For administration of recombinant murine IL-21 (rIL-21) (PEPROTECH), mice were inoculated intranasally with 0.5 μg rIL-21 in 20 μl PBS at 1 day before infection and days 0, 2, 4, and 6, and the control group was given 20 μl sterile PBS in the same schedule. Mice were monitored daily for body weight changes and euthanized for analysis at designated time points after infection.

Zeng J., Xu Y., Tan L., Zha X., Yang S., Zhang H., Tuo Y., Sun R., Niu W., Pang G., Sun L, & Bai H. (2022). IL-21/IL-21R Regulates the Neutrophil-Mediated Pathologic Immune Response during Chlamydial Respiratory Infection. Mediators of Inflammation, 2022, 4322092.

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Purification and Culture of Murine Germinal Center B Cells

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Splenic naive B cells were purified with the MagniSort Streptavidin Negative Selection Beads (eBioscience) following the manufacturer’s instructions, using TCRb-Bio (BioLegend, 109204), CD11b-Bio (Biolegend, 101204) and Ter119-Bio (Biolegend, 116204). iGC B cell culture was performed following (Nojima et al., 2011 (link)). Briefly, B cells were plated (5 × 10⁶ cells per 10 cm dish) on mitomycin C (Sigma, M0305)-treated 40LB feeder cells (3 × 10⁶ cells per 10 cm dish) in 40 mL B cell medium: DMEM (Sigma, WHMISDZB)) supplemented with 10% (v/v) FBS (GIBCO, 10270-106), 2 mM L-glutamine (Sigma, G7513), 10 mM HEPES (LONZA, BE17-737E), 1 mM sodium pyruvate (GIBCO, 13360-039), 1 x non-essential amino acids (GIBCO, 11140-035), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma, 0781), 50 μM β-mercaptoethanol (Sigma, M3148) and 10 ng/ml rIL-4 (Peprotech, 214-14); 30 mL of medium were changed on day 3. On day 4, iGC B cells were harvested, analyzed and 1,5 × 10⁶ cells replated per 10 cm dish onto a new Mitomycin C-treated feeder layer in B cell medium supplemented with either 10 ng/ml rIL-4 or 10 ng/ml rIL-21 (Peprotech, 210-21); 30 mL of medium were changed on day 7 and the final analysis was performed on day 8. The analysis included cell count with the trypan blue exclusion method using a hemocytometer (Neubauer) and flow cytometric analysis, as described above.

Olson W.J., Jakic B., Labi V., Schoeler K., Kind M., Klepsch V., Baier G, & Hermann-Kleiter N. (2019). Orphan Nuclear Receptor NR2F6 Suppresses T Follicular Helper Cell Accumulation through Regulation of IL-21. Cell Reports, 28(11), 2878-2891.e5.

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Splenic B Cell Culture and Expansion

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Purified splenic naive B cells and splenic EYFP⁺ memory B cells (5 × 10⁴ cells per well; Nojima et al., 2011 (link)) were cultured in 6-well plates in the presence of 3T3 40LB cells (3 × 10⁵ cells per well) that had been pretreated with mitomycin C (1 mg/ml during 3 h; Sigma) in 8 ml RPMI-1640 medium supplemented with 10% FCS, 5.5 × 10⁻⁵ M 2-mercaptoethanol, 10 mM Hepes, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO). rIL-4 (1 ng/ml; Peprotech) was added to the primary culture for 4 d. On day 4, the cells were replated (5 × 10⁵ cells) onto a new feeder layer treated with mitomycin C (3 × 10⁶ cells) in a 10-cm tissue culture dish and cultured in 40 ml of medium supplemented with rIL-21 (10 ng/ml, Peprotech) for 6 d. Cells were cultured in a humidified atmosphere at 37°C with 5% CO₂. At day 10, 38 ml of supernatant were collected and concentrated using an Amicon Ultra 100000 MCWO (Millipore).

Le Gallou S., Zhou Z., Thai L.H., Fritzen R., de los Aires A.V., Mégret J., Yu P., Kitamura D., Bille E., Tros F., Nassif X., Charbit A., Weller S., Weill J.C, & Reynaud C.A. (2018). A splenic IgM memory subset with antibacterial specificities is sustained from persistent mucosal responses. The Journal of Experimental Medicine, 215(8), 2035-2053.

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Functional Evaluation of B-Cell Interactions

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The isolated CXCR5⁺CD4⁺ or CXCR5⁺CD4⁻ T cells from five treatment-naïve PBC patients or four healthy controls were incubated with allogeneic CD19+ B cells from healthy controls in a 96-well plate, at a ratio of 1:1 in the presence of SEB (100ng/ml) in RPMI 1640 complete medium containing 10% FCS only or with rIL-21R-Fc (10 μg/ml; R&D Systems, Minneapolis, MN) or rIL-21 (20 ng/ml; Peprotech, Rocky Hill, NJ, USA) (30 (link), 31 (link)). After 7–8 days of culture, cells were harvested to evaluate surface phenotypes and supernatants were collected to detect IgM and IgG (32 (link)).

Wang L., Sun Y., Zhang Z., Jia Y., Zou Z., Ding J., Li Y., Xu X., Jin L., Yang T., Li Z., Sun Y., Zhang J.Y., Lv S., Chen L., Li B., Gershwin M.E, & Wang F.S. (2015). CXCR5+ CD4+ T Follicular Helper Cells Participate in the Pathogenesis of Primary Biliary Cirrhosis. Hepatology (Baltimore, Md.), 61(2), 627-638.

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Isolation and Differentiation of Splenic Naive B Cells

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Splenic naive B cells were purified with the MagniSort Streptavidin Negative Selection Beads (eBioscience) following the manufacturer’s instructions, using TCRb-Bio (BioLegend, 109204), CD11b-Bio (Biolegend, 101204) and Ter119-Bio (Biolegend, 116204). iGC B cell culture was performed following (Nojima et al., 2011 ). Briefly, B cells were plated (5 × 10⁶ cells per 10 cm dish) on mitomycin C (Sigma, M0305)-treated 40LB feeder cells (3 × 10⁶ cells per 10 cm dish) in 40 mL B cell medium: DMEM (Sigma, WHMISDZB)) supplemented with 10% (v/v) FBS (GIBCO, 10270-106), 2 mM L-glutamine (Sigma, G7513), 10 mM HEPES (LONZA, BE17-737E), 1 mM sodium pyruvate (GIBCO, 13360-039), 1 x non-essential amino acids (GIBCO, 11140-035), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma, 0781), 50 μM β-mercaptoethanol (Sigma, M3148) and 10 ng/ml rIL-4 (Peprotech, 214-14); 30 mL of medium were changed on day 3. On day 4, iGC B cells were harvested, analyzed and 1,5 × 10⁶ cells replated per 10 cm dish onto a new mitomycin C-treated feeder layer in B cell medium supplemented with either 10 ng/ml rIL-4 or 10 ng/ml rIL-21 (Peprotech, 210-21); 30 mL of medium were changed on day 7 and the final analysis was performed on day 8. The analysis included cell count with the trypan blue exclusion method using a hemocytometer (Neubauer) and flow cytometric analysis, as described above.

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Proliferation of Intestinal PP B Cells

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About 800 sorted CD11b⁺IgA⁺ PP B cells and 800 sorted CD11b⁻IgA⁺ PP B cells were stained with a CellTrace Violet Proliferation Kit (Invitrogen, USA). Since the number of sorted CD11b⁺IgA⁺ PP B cells was very small, 5000 CD11b⁻IgA⁺ PP B cells and 5 × 10⁴ naive spleen B cells [negatively sorted by a B cell isolation kit (Miltenyi Biotec, Germany)] were prepared as positive controls. To monitor cell proliferation, the sorted CD11b⁻IgA⁺ PP B cells, CD11b⁺IgA⁺ PP B cells and naive spleen B cells were seeded in a 6-well tissue culture dish in the presence of 40LB cells that had been pre-treated with mitomycin C to inhibit the growth. Cells were cultured in RPMI-1640 medium (Wako, Japan) [containing 10% FCS, 5.5 × 10⁻⁵ M 2-Mercaptoethanol (ME) (Nacalai, Japan), 10 mM HEPES (Nacalai, Japan)] at 37°C with 5% CO₂ for 3 days. Sorted CD11b⁺IgA⁺ PP B cells and CD11b⁻IgA⁺ PP B cells were cultured with rIL-21 (10 ng ml⁻¹; PeproTech, USA) and naive spleen B cells were cultured with rIL-4 (1 ng ml⁻¹; Biolegend, USA). Flow cytometry analysis was performed to detect CellTrace Violet from day 0 to day 3 with the iGB culture system. CD11b expression of cultured cells was analyzed on day 0 and day 1 by flow cytometry with a Cell Sorter SH800 (SONY, Japan) and a Spectral Cell Analyzer SA3800 (SONY, Japan).

Gao P., Adachi T., Okai S., Morita N., Kitamura D, & Shinkura R. (2021). Integrin CD11b provides a new marker of pre-germinal center IgA+ B cells in murine Peyer’s patches. International Immunology, 34(5), 249-262.

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Immunophenotyping of pSTAT3 activation

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For the detection of pSTAT3, cells were stained for lineage markers in PBS for 30 minutes, then stimulated for 30 minutes with 10 ng/ml rIL-21 (eBioscience). Surface marker and pSTAT3 staining was performed following fixation with 4% paraformaldehyde and permeabilization with 100% methanol. pSTAT3 staining was achieved with Phospho-Stat3 (Y705) antibody (Cell Signaling)..

Poholek C.H., Dulson S.J., Zajac A.J, & Harrington L.E. (2019). Interleukin-21 Controls ILC3 Cytokine Production and Promotes a Protective Phenotype in a Mouse Model of Colitis. ImmunoHorizons, 3(6), 194-202.

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IL-7, SCF, and IL-21 Regulate ILC3s

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ILC3s were FACS sorted from SI lamina propria and 1×104 cells/well were plated in 100 μl complete media supplemented with 10 ng/ml rIL-7 (Biolegend), 10 ng/ml stem cell factor (Biolegend), and +/− 50 ng/ml rIL-21 (eBioscience). Cells were cultured overnight, and RNA was extracted for real-time PCR as described below.

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