Ril 2

Manufactured by Merck Group

Sourced in United States, New Zealand, China

RIL-2 is a laboratory equipment used for measurement and analysis purposes. It functions as an integral part of research and testing workflows in various scientific and medical fields.

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Close spelling variants of this product

Human rIL-2 (2 citations) Recombinant IL-2 (rIL-2) (2 citations)

10 protocols using ril 2

Murine Splenocyte Activation for Cytotoxicity

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Spleens were harvested from 16–20 week old male pmel-1 mice, mechanically disrupted, filtered through a 70 μm filter (BD Biosciences), washed with PBS, and centrifuged at 450 × g for 5 minutes. The pellet was re-suspended in ACK lysis buffer for 2 minutes to lyse the red blood cells, filtered through a 70 μm filter, washed with PBS, and centrifuged at 450 × g for 5 minutes. The splenocytes were then cultured in one of two ways, 1) with 0.1 ug/mL gp100_25–33 peptide and 30 IU/mL rIL-2 (Sigma-Aldrich) for 5 days, 2) with CD3/CD28 beads (Thermo Fisher) and 30 IU/mL rIL-2 for 5 days, in RPMI 1640 containing 10% FBS, 2-mercaptoethanol, nonessential amino acids, sodium pyruvate, and penicillin/streptomycin at 37°C in 5% CO₂. Flow cytometric analysis confirmed a CD8⁺ T cell effector phenotype prior to use in the cytotoxicity assay.

Hoffend N.C., Magner W.J, & Tomasi T.B. (2016). The modulation of Dicer regulates tumor immunogenicity in melanoma. Oncotarget, 7(30), 47663-47673.

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Enrichment and Analysis of Antigen-Specific T Cells

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CD4⁻ cells that had been MACS-purified from thymocytes and splenocytes were incubated with PE-labeled H60-tetramers and then with anti-PE antibody-conjugated magnetic beads (Miltenyi Biotech, Auburn, CA, USA) for enrichment of the antigen-specific T cells as described previously^{32 (link),59 (link),60 (link)}. These MACS-enriched cells were stained with H60-tetramers and antibodies for flow cytometric analysis. Alternatively, the CD4⁻ cells were subjected to MLC for 7 days and then re-stimulated weekly with irradiated (2000 cGy) Con-H60 splenocyte feeder cells in DMEM containing 5% FBS (Hyclone, Logan, UT, USA) and rIL-2 (50 U/ml; Sigma-Aldrich) as previously described^{27 (link)}.

Ju J.M., Jung M.H., Nam G., Kim W., Oh S., Kim H.D., Kim J.Y., Chang J., Lee S.H., Park G.S., Min C.K., Lee D.S., Kim M.G., Choi K, & Choi E.Y. (2018). Escape from thymic deletion and anti-leukemic effects of T cells specific for hematopoietic cell-restricted antigen. Nature Communications, 9, 225.

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Telomerase Activity in T-Cell Subtypes

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For determination of telomerase activity naïve and memory CD4⁺ and CD8⁺ T-cells were isolated and cultured in advanced RPMI 1640 (GIBCO) in the presence of 30 U/mL rIL-2 (Sigma, Vienna, Austria) and CD3/CD28 Dynabeads (bead:cell ratio 1:1; Invitrogen). After 3 and 6 days, 2 × 10⁵ cells were harvested and telomerase activity was determined using TeloTAGGG Telomerase PCR ELISAplus kit (Roche) according to ‘Telomeric Repeat Amplification Protocols (TRAP)’.21 (link)

Fessler J., Raicht A., Husic R., Ficjan A., Duftner C., Schwinger W., Dejaco C, & Schirmer M. (2015). Premature senescence of T-cell subsets in axial spondyloarthritis. Annals of the Rheumatic Diseases, 75(4), 748-754.

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SARS-CoV-2 Spike Protein Peptide Stimulation

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PBMCs were isolated from heparinized whole blood using Ficoll–Paque (GE Healthcare, Singapore). Then, PBMCs from I-I-I were treated with the peptide pool containing 384 15-mer peptides spanning the antigen region of spike (S) protein (250 nM per peptide) in presence of 10 U/mL recombinant interleukin-2 (rIL-2) and 1 μM GolgiPlug (BD Biosciences, San Diego, CA) for 16 h at 37 °C, 5% CO₂. PBMCs from BA.5 infection were treated with the peptide pool containing 487 15-mer peptides spanning the antigen region of spike (S), membrane (M), nucleocapsid (N), and envelope (E) proteins (250 nM per peptide) in presence of 10 U/mL rIL-2 and 1 μM GolgiPlug (BD Biosciences, San Diego, CA) for 16 h at 37 °C, 5% CO₂. RPMI 1640 medium (Gibco, Waltham, MA) supplemented with 10% heat-inactivated FBS (Biological Industries, Israel Beit-Haemek), 100 U/mL penicillin (Gibco, Waltham, MA), 0.1 mg/mL streptomycin (Gibco, Waltham, MA), 10 U/mL rIL-2, and 0.01% DMSO (Sigma, Saint Louis, MO) was used as subtraction control.

Cui T., Su X., Sun J., Liu S., Huang M., Li W., Luo C., Cheng L., Wei R., Song T., Sun X., Luo Q., Li J., Su J., Deng S., Zhao J., Zhao Z., Zhong N, & Wang Z. (2023). Dynamic immune landscape in vaccinated-BA.5-XBB.1.9.1 reinfections revealed a 5-month protection-duration against XBB infection and a shift in immune imprinting. eBioMedicine, 99, 104903.

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T cell Isolation and Functional Analysis

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Reagent used were: human lymphocyte isolation medium (Sigma, USA); Pan T Cell Isolation Kit (Milteni, Germany); 10% foetal bovine serum (FBS) (Hyclone, New Zealand); RPMI 1640 medium (Hyclone, New Zealand); rIL-2 (Sigma, US); FITC-CD3 (Becton, Dickinson and Company, USA); Trizol (Life Technologies, USA); Reverse transcription kit (Thermo Fisher, USA); RT-PCR kit (Qiagen, Germany); RIPA Lysis Buffer (Thermo Fisher, USA); Protease inhibitor (Thermo, USA); BCA Protein Assay Kit (Thermo, USA); SDS-PAGE (BIO-RAD, USA); anti-human monoclonal antibodies against NFATc1, IL-6, TNF-α, and β-actin, and goat anti-rabbit secondary antibodies (Abcam, UK); BCIP/NBT chromogenic reagent (Invitrogen, USA); telmisartan (Boehringer-Ingelheim, Germany); CsA (Novartis, Switzerland); VIVIT (Sigma, USA); 4-AP (Sigma, USA).

Huang S.S., He S.L, & Zhang Y.M. (2016). The effects of telmisartan on the nuclear factor of activated T lymphocytes signalling pathway in hypertensive patients. Journal of the Renin-Angiotensin-Aldosterone System: JRAAS, 17(2), 1470320316655005.

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Comprehensive Immune Cell Phenotyping

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Surface markers on cells were stained with Abs in PBS with 2% bovine growth serum (FACS buffer) at 4°C for 20 min. For T_FH staining, cells were stained with purified CXCR5 for 1 h in FACS buffer + 0.5% BSA + 2% normal mouse serum (NMS), followed by biotin-goat anti-rat IgG for 30 min in FACS buffer + 2% NMS, and then streptavidin and other surface-staining Abs for 30 min in FACS buffer + 2% NMS at 4°C. For intracellular cytokine/molecule detection, splenocytes were restimulated ex vivo with 2.5μg/ml gp150_67–83 peptide (LSNNNPTTIMRPPVAQN) or 50ng/ml PMA (Sigma-Aldrich, Milwaukee, WI) and 1μg/ml ionomycin calcium salt from Streptomyces conglobatus (Sigma-Aldrich) in complete medium with 10U/ml rIL-2 and 10μg/ml brefeldin A (Sigma-Aldrich) at 37°C for 5h. Subsequently, cells were stained with Abs against surface markers for 20 min at 4°C, followed by fixation with 1% formaldehyde at 4°C for 20 min, and then stained with Abs against IFN-γ, TNF-α, IL-2 or GzmB in 0.5% saponin solution at 4°C for 30 min. For transcription factor detection, Foxp3/Transcription Factor Staining Buffer Set (eBioscience) was used.

Hu Z., Blackman M.A., Kaye K.M, & Usherwood E.J. (2015). Functional Heterogeneity in the CD4+ T Cell Response to Murine γ-Herpesvirus 68. Journal of immunology (Baltimore, Md. : 1950), 194(6), 2746-2756.

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Decitabine Modulates Activated CD4+ T Cells

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Mouse CD4⁺ T cells were purified from splenocytes of C57BL/6J using CD4⁺ T Cell Isolation Kit (Miltenyi, Cat#130-104-454) according to the manufacturer’s guide. CD4⁺ T cells were activated in vitro with plate-bound 2 μg/ml anti-CD3 (Biolegend, Cat#100340) and 2 μg/ml anti-CD28 (Biolegend, Cat#102116) and recombinant IL-2 (cyagen, Cat#MEILP-0201) for 24 h. Human CD4⁺ T cells were isolated from peripheral blood mononuclear cells of healthy donors and cancer patients using CD4⁺ T cell Isolation (Miltenyi, Cat#130-045-101). The sorted human CD4⁺ T cells were activated with plate-bound anti-CD3 antibody (Takara, Cat#T210) and rIL-2 (cyagen, Cat#HEILP-0201) for 24 h. Activated CD4⁺ T cells were treated with PBS (CON) or decitabine (10 nM or indicated concentrations, Sigma-Aldrich, Cat#A3656) plus rIL-2 for 3 days. After decitabine treatment, CD4⁺ T cells were analyzed by flow cytometry or adoptively transferred into tumor-bearing mice.

Li X., Dong L., Liu J., Wang C., Zhang Y., Mei Q., Han W., Xie P, & Nie J. (2021). Low-Dose Decitabine Augments the Activation and Anti-Tumor Immune Response of IFN-γ+ CD4+ T Cells Through Enhancing IκBα Degradation and NF-κB Activation. Frontiers in Cell and Developmental Biology, 9, 647713.

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Cytokine-Induced Killer Cell Expansion

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For CIK induction, 2×10⁶ cells/ml were incubated in IMDM containing 1,000 U/mL of recombinant human IFN-γ (rIFN-γ, Sigma-Aldrich, St. Louis, MO, USA; SRP3058) for 24 hours, followed by the addition of 400 ng/mL CD3 monoclonal antibody (mAb; T&L Biotechnology, Beijing, China, TL-101) and 300 U/mL recombinant human IL-2 (rIL-2; Sigma-Aldrich; IL002) at 37 °C with 5% CO₂ for 14 days. Every 3 days, half of the medium was replaced with fresh medium containing 300 U/mL rIL-2. The phenotype of CIK cells was determined, and CD56-positive cells were isolated. CIK cells were enriched after expansion by immunomagnetic selection using CD56 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's protocol. Fluorescein-5-isothiocyanate (FITC)-human leukocyte antigen class I (HLA-I) antibody (Thermo Fisher Scientific, Waltham, MA, USA) was added to the CIK cells and incubated at 4 °C in the dark for 30 minutes. The HLA-I⁺ CIK cells were analyzed using a FACSCalibur flow cytometer (BD, Heidelberg, Germany).

Jiang R., Zhang Z., Liao X., Huang L., Liao Y, & Deng W. (2021). Combination of oncolytic adenovirus ZD55 harboring TRAIL-IETD-MnSOD and cytokine-induced killer cells against lung cancer. Annals of Translational Medicine, 9(20), 1527.

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PBMC Stimulation and Rosuvastatin Treatment

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PBMCs from each group were cultured and proliferated with RPMI-1640 culture medium containing 10% FBS (both from Thermo Fisher Scientific, Inc.) and 50 U/ml rIL-2 (Sigma-Aldrich; Merck KGaA) for 24 h in vitro. Then, the PBMCs were harvested and equally divided into three portions. One portion was defined as the untreated group (0 h) and collected directly for RNA and protein extraction. The other two portions were sequentially cultured with Rosuvastatin for another 12 and 24 h, respectively, and were defined as the 12 and 24-h groups. Rosuvastatin (Sigma-Aldrich; Merck KGaA) was dissolved in DMSO at a stock solution of 20 mM and stored in aliquots at −20°C as previously described (19 (link)).

Zhu J., Wu S., Hu S., Li H., Li M., Geng X, & Wang H. (2019). NLRP3 inflammasome expression in peripheral blood monocytes of coronary heart disease patients and its modulation by rosuvastatin. Molecular Medicine Reports, 20(2), 1826-1836.

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Rat Acclimation and IL-2 Treatment

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Male Han Wistar rats (Crl:Wi[Han]) aged 8 weeks were purchased from Charles River Laboratories (Raleigh, NC) and were acclimated 2 weeks before dosing. Animals were randomized by weight and assigned to either vehicle or rIL-2 (Proleukin 1 ; Sigma-Aldrich, St. Louis, MO) groups and single housed in polycarbonate solid-bottom cages. Upon study initiation, the rats were 305 + 13 g (mean + standard deviation). Animal identification and conditions of housing, acclimatization, environment, diet, and water were in accordance with facility standard operating procedures. All animal procedures were conducted in an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility under an Institutional Animal Care and Use Committee approval protocol.

Keirstead N.D., Bertinetti-Lapatki C., Knapp D., Albassam M., Hughes V., Hong F., Roth A.B, & Mikaelian I. (2015). Temporal Patterns of Novel Circulating Biomarkers in IL-2-mediated Vascular Injury in the Rat. Toxicologic pathology, 43(7).

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