Frozen PBMCs were thawed and rested at 2-3x106 cells/mL in TCM containing IL-2 (50 U/mL; BioLegend) and IL-15 (5 ng/mL; BioLegend) overnight at 37 °C, 5% CO2. PBMCs then were activated for 2 days with anti-human CD3/CD28 magnetic dynabeads (Thermo Fisher Scientific) at a beads to cells ratio of 1:1 in IL-2 (200 U/mL; BioLegend) and IL-15 (5 ng/mL; BioLegend) supplemented TCM. Activated PBMC cells (5-10 x106) were harvested and electroporated with RNP mixture and HDR DNA templates (2.5 µg) using P3 Primary Cell 4D-Nucleofector™ X Kit S (Lonza) and a 4D Nucleofector X unit (Lonza) using EO115 electroporation program following the manufacturer’s protocol. Electroporated cells were seeded into a 24 well plate at a density of 5-10x106 cells/mL in TCM containing IL-2 (200 U/mL; BioLegend). Mouse TCRβ-expressing T cells were sorted 3-5 days after electroporation and expanded with mixed lymphocyte reaction. The expanded TCR-transgenic T cells were subject to subsequent functional assays. When co-culturing with K562-EV/CD1a or GAS-infected K562-EV/CD1a cells for 4 hrs, expanded TCR-transgenic T cells were supplied with IL-6 (5 ng/mL; BioLegend), TNF-α (5 ng/mL; BioLegend), IL-2 (25 U/mL; BioLegend) and anti-CD3 (OKT3; 5 ng/ml; BioLegend) or IL-12 (1 ng/mL; BioLegend), IL-18 (1 ng/mL; BioLegend) and IL-2 (25 U/mL; BioLegend) to support CD1a-dependent cytokine production.
Interleukin 2 (il 2)
Manufactured by BioLegend
Sourced in United States, Switzerland, Germany, Canada
IL-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells. It is an essential component in the immune system, involved in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by BioLegend (29 mentions)
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Interleukin 6 (il 6), by BioLegend (20 mentions)
Il 12, by BioLegend (17 mentions)
Il 15, by BioLegend (15 mentions)
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Anti cd28, by BioLegend (10 mentions)
Tgf β, by BioLegend (10 mentions)
Anti il 4, by BioLegend (10 mentions)
Il 10, by BioLegend (10 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (10 mentions)
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Rpmi 1640, by Thermo Fisher Scientific (8 mentions)
Il 23, by BioLegend (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin, by Thermo Fisher Scientific (8 mentions)
Il 1β, by BioLegend (7 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
237 protocols using interleukin 2 (il 2)
1
Genome Editing of Primary T Cells
2
Expansion and Maintenance of Cell Types
3
In Vitro T Cell Polarization Assay
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Purified naïve CD4+ T cells were cultured in 24-well (4 x 105 cells per well), 48-well (2 x 105 cells per well) or 96-well (1 x 105 cells per well) plates coated with 5 μg/mL anti-CD3ϵ antibody (145-2C11; Biolegend) in IMDM medium (12440-061; Invitrogen) containing 1 μg/mL anti-CD28 antibody (37.51; Biolegend), 10% FBS, 1x streptomycin-penicillin (containing 100 U/mL penicillin and 100 μg/mL streptomycin, P4333; Sigma), and 55 μM β-mercaptoethanol (20985-023; Invitrogen, Waltham, MA, USA). In addition, the following cytokines and antibodies were added in each polarizing condition: 20 ng/mL IL-2 (570402; Biolegend), 1 μg/mL anti-IFN-γ (XMG1.2; Biolegend), and 1 μg/mL anti-IL-4 (11B11; Biolegend) for Th0; 20 ng/mL IL-2, 20 ng/mL IL-12 (577002; Biolegend), and 1 μg/mL anti-IL-4 for Th1; 20 ng/mL IL-2, 100 ng/mL IL-4 (574306; Biolegend), and 1 μg/mL anti-IFN-γ for Th2; 20 ng/mL IL-6 (575706; Biolegend), and 3 ng/mL TGF-β1 (100-21C; PeproTech, Cranbury, NJ, USA) for Th17. Polarized cells were harvested for further analysis at the indicated time points.
4
Differentiation of Induced Regulatory T Cells
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To investigate the differentiation of iTreg, CD4+ naïve T cells were purified using a commercially available isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, Germany) and stimulated with plate-coated anti-CD3 antibody (1 mg/ml, Biolegend, USA) and soluble anti-CD28 antibody (1 mg/ml, Biolegend, USA) in X-VIVO15 medium (Lonza, Switzerland). Three days after plating, TGF-b (1 ng/ml, Biolegend, USA) and interleukin 2 (IL-2, 50 U/ml, Biolegend, USA) were added in the culture, and the cells were continued to cultivate for 2 days. According to different experimental purposes, reagents were added together with the polarization cytokines, including methyl-b-cyclodextrin-cholesterol (cholesterol, 10–20 mg/ml, Sigma, USA), dimethyloxalylglycine (DMOG, 100 mM, MCE, USA), PX-478 (10 mM, MCE, USA), MT (10 mM, Sigma, USA) and IL-1b (100 ng/ml, Biolegend, USA).
5
Regulatory T Cell-Mediated CD8+ T Cell Suppression
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BMDMs were cocultured with naïve CD4+ T cells and differentiated into Tregs in the presence 0.5 μg/mL (anti-CD3 BioLegend), 1ng/mL TGFβ (BioLegend) and 1ng/mL IL-2 (BioLegend) for 72 hr. Next, expanded CD4+ T cells were cocultured at different ratios with CFSE/CTV labeled CD8+ T cells and stimulated with Mouse T-Activator CD3/CD28 Dynabeads at 2 cells per bead ratio and 0.5ng/mL IL-2 (BioLegend). Cell culture media consisted with RPMI 1640, supplemented with 10% heat-inactivated charcoal-stripped FBS, 50 µM β-mercaptoethanol, 1% sodium pyruvate, 1% nonessential amino acids, 1% penicillin/ streptomycin, and 1% Glutamax. CD8+ T cells were isolated from wildtype mice spleens using CD8a+ T Cell Isolation Kit (130-104-075) from Miltenyi Biotec according to the manufacturer’s instructions. Flow cytometric analysis of suppressive cultures was preformed after 72 h. Beads were removed using DynaMag magnet (Invitrogen) prior to flow cytometry staining and analysis.
6
Profiling Cytokine Secretion in ILC2s
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Bone marrow cells (5 × 105 cells per well) were plated in a round bottom 96-well plate and co-cultured with IL-2, IL-7, IL-33 at 10 ng/ml (Biolegend), and recombinant parasite-derived proteins, at 37°C, 5% CO2 for 5 days. Supernatants were collected and analysed by IL-5, IL-6 and IL-13 ELISA according to manufacturer’s instructions (Thermo Fisher). Where biotinylated constructs were used to assess binding to bone marrow ILC2s, BALB/c or ST2-deficient bone marrow cells were cultured for 5 days in IL-2, IL-7 and IL-25 at 10 ng/ml (Biolegend) to support ILC2 proliferation, prior to incubation for 20 min at 4°C with HES and recombinant proteins, followed by biotinylated recombinant proteins for a further 20 min, as indicated, and avidin-PE to detect binding.
7
Polarized CD4+ T cell generation
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Polarized CD4+ effector T cells were generated by activating purified HD naïve CD4 T cells with plate-bound anti-CD3 (UCHT1) and anti-CD28 (CD28.2) (both 5 μg/ml, Biolegend) in the presence of IL-2 (50 U/ml), IL-12 (1 ng/ml) and anti-IL4 (10 μg/ml) (Th1 conditions) or IL-2 (50 U/ml), IL-4 (20 ng/ml), anti-IL12 (10 μg/ml) and anti-IFNγ (10 μg/ml) (Th2 conditions). Cells were transferred into fresh media on day 3 and IL-2 was added, as needed. Cells were re-activated every 7 days using the same cultures conditions for 3 rounds of polarization. All cytokines and Abs except IL-2 (Peprotech) were purchased from R and D and T cell polarizing media contained Iscove’s DMEM supplemented with penicillin (200 μg/ml), streptomycin (200 μg/ml), gentamicin (40 μg/ml), 10% FBS and 5% human serum blood type AB.
8
Activation of CD1a-autoreactive T-cells
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EV‐K562 and CD1a‐K562 cells were pulsed with 10 μg recombinant‐AOAH overnight at 37°C and 5% CO2. Following the overnight culture, K562 cells were washed to remove excess AOAH. K562 (2 × 105) cells were cocultured with 1–5 × 105 CD1a‐autoreactive T‐cell clones for 4 h. IFN‐Ɣ‐producing T‐cell clone culture was supplied with IL‐12 (1 ng/mL, BioLegend), IL‐18 (1 ng/mL, BioLegend), and IL‐2 (25 U/mL, BioLegend); and IL‐22‐producing T‐cell clone culture were supplied with IL‐6 (5 ng/mL, BioLegend), TNF‐α (5 ng/mL, BioLegend), and IL‐2 (25 U/mL, BioLegend). Activation of T‐cell clones was accessed by cytokine production of T cells using Secretion Assay (Miltenyi Biotec) following the manufacturer's instructions. Data were collected using a LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc.).
9
Isolation and Activation of Regulatory T Cells
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Live tTregs (CD3+CD4+CD25+Foxp3GFP+Nrp1+ ) and pTregs (CD3+CD4+CD25+Foxp3GFP+Nrp1−) were isolated from the spleens of Foxp3GFP and PD-L2KO Foxp3GFP mice. 0.2–1.5 × 105 cells were further stimulated in U-bottom 96-well plates with a 1:1 ratio of Dynabeads Mouse T-activator CD3/CD28 (Gibco), and in some experiments 20 ng/mL IL-2 (Biolegend) and 5 ng/mL TGF-β (ebioscience) in cRPMI. Cells were incubated for up to 4 days at 37 °C and analyzed for the selected readouts. In some experiments, 2 mM methyl pyruvate (Sigma) was added to the cultures. When indicated, CD3–CD4+CD44−CD62L+ naive T cells were isolated from BALB/c splenocytes. For experiments involving iTregs, 1–10 × 105 naive T cells were incubated in U-bottom 96-well plates or flat-bottom 48-well plates with a 1:1 ratio of Dynabeads Mouse T-activator CD3/CD28 (Thermofisher), 20 ng/mL IL-2 (Biolegend) and 5 ng/mL TGF-β (ebioscience) in cRPMI. When indicated, 5 µg/mL of rmPD-L2Fc or corresponding rmIgG1Fc control (both R&D Systems) were coated in the wells for 30–60 min at 37 C before culture. For the BMDC/Treg co-cultures, OVA-specific KJ126+CD4+CD44−CD62L+ naive T cells were isolated from DO11.10 splenocytes and used as described below. When indicated, 106 WT Foxp3GFP iTregs were adoptively transferred to WT BALB/c or PD-L2KO mice.
10
Tumor-Induced Modulation of T Cell Activation
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CD3+ T cells were isolated from peripheral blood mononuclear cells by using a pan T cell isolation cocktail kit (Miltenyi Biotec) and MACS. CD3+ T cells (1 × 105 cells) labeled with carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) were co-cultured either with 0.2 × 105 cells of mDC only or mDC-MDA-MB-231 for 4 days, and the percentage of divided cells was measured by flow cytometry. For the activation-induced cell death (AICD) assay, CD3+ T cells that had been previously co-cultured either with mDC only or mDC-MDA231 were purified and allowed to rest for 1 day in the presence of 50 units/ml IL-2 (BioLegend). Then, the CD3+ T cells were stimulated with plate-bound anti-CD3 (5 μg/ml, BioLegend) and soluble anti-CD28 (1 μg/ml, BioLegend) antibodies in the presence of 50 units/ml IL-2 for additional 2 days. The stimulated CD3+ T cells were stained with PI (BD Bioscience) and annexin V (BD Bioscience), and analyzed by flow cytometry.
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