Naive CD4+CD25− T cells were purified from LNs and spleen of WT C57BL/6, CD4CrePkm2fl/fl or control littermate (CD4Cre and Pkm2fl/fl) mice with the untouched CD4 T cell isolation kit (Miltenyi Biotec) and a biotinylated CD25 monoclonal antibody (eBioscience) by using an AutoMACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer’s protocol. Purified cells were activated with soluble anti-CD3ε:CD28 (both 1 µg/ml; BD Biosciences) on U-bottomed plates (105/well). Skewing conditions were as follows: Th17, 2.5 ng/ml rhTGF-β1 (eBioscience) plus 20 ng/ml rmIL-6 (R&D Systems) with or without 20 ng/ml rmIL-23 (R&D Systems); Th1, rmIL-12, and rmIL-2 (both 20 ng/ml; R&D Systems); Th2, anti-IFN-γ (10 µg/ml), rmIL-4, and rmIL-2 (both 20 ng/ml; R&D Systems). For iT reg cell polarization, naive T cells were cultured with plate-bound CD3ε:CD28 (both 1 µg/ml; BD Biosciences) in the presence of 1 ng/ml rhTGF-β1 (eBioscience). When indicated, 0.1 µM rapamycin (Cayman Chemical), 100 µM TEPP-46 (Millipore), or 2 µM Stattic (Tocris) was used.
Rmil 2
Manufactured by R&D Systems
Sourced in United States
RmIL-2 is a recombinant mouse interleukin-2 protein. Interleukin-2 is a cytokine that plays a central role in the activation and proliferation of T cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Rmil 12, by R&D Systems (5 mentions)
Rmil 6, by R&D Systems (4 mentions)
Rmil 7, by R&D Systems (4 mentions)
Mouse ifn γ enzyme linked immunospot elispot assay, by BD (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Rmil 23, by R&D Systems (3 mentions)
Rhtgf β1, by Thermo Fisher Scientific (3 mentions)
Automacs magnetic cell sorter, by Miltenyi Biotec (3 mentions)
Rmil 33, by R&D Systems (3 mentions)
Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (3 mentions)
Celltrace violet, by Thermo Fisher Scientific (3 mentions)
Rmil 33, by BioLegend (2 mentions)
Anti cd3ε cd28, by BD (2 mentions)
Rhtgf β1, by R&D Systems (2 mentions)
C di amp, by InvivoGen (2 mentions)
C di gmp, by InvivoGen (2 mentions)
Rmil 1β, by R&D Systems (2 mentions)
25 protocols using rmil 2
1
T cell Differentiation and Modulation
2
Labeling T cells for in vivo tracking
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Single T cell suspensions derived from erythrocyte-depleted spleen cell suspensions of OT-1 and B6 mice (155 mM NH4Cl, 10 mM KHCO3, 0,1 mM EDTA) by activation of 2 × 106 cells/ml splenocytes in T cell medium for 48 hours. OT-1 T cell suspensions were supplemented with 2 µg/ml 50 U/ml rm IL-2 (R&D) and 0.1 µg/ml CD28 (R&D), B6 T cell were activated in CD3 coated cell culture flasks (1 µg/ml, R&D) supplemented with 50 U/ml rmIL-2 and 0.1 µg/ml CD28. Next, cells were expanded at a density of 1 × 106 cells/ml in T cell medium supplemented with 40 ng/ml rmIL-15 (R&D) for additional 3 days. On day 5 after isolation, cells were labeled with 10 µM Xenolight DiR (Perkin Elmer) in 1x PBS at a concentration of 1 × 106 cells/ml for 15 minutes at 37 °C, with 24 µM VivoTag-S 750 (Perkin Elmer) in T cell medium at a concentration of 4 × 106 cells/ml for 30 minutes at 37 °C or double labeled with both dyes. After washing twice with 1x PBS, labeled T cells were incubated for 2 hours in T cell medium with 40 ng/ml IL-15 to recover. Subsequently, labeled cells were transferred into animals or further analysed in vitro. Labeling conditions for VivoTag-750 were analysed using full medium and serum-free medium and the same conditions as described earlier.
3
T-cell Differentiation Protocol
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All the cultures of T cells used RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 100 IU ml−1 penicillin, 100 μg ml−1 streptomycin, 10 mM HEPES (Gibco) and 5 mM β-mercaptoethanol (Gibco). The naive CD4+CD25−Foxp3gfp-CD62Lhi T cells were activated with plate-bound anti-CD3 (5 μg ml−1; 145-2C11; BD Biosciences) plus soluble anti-CD28 (2 μg ml−1; 37.51; BD Biosciences). TH1-cell differentiation conditions included 10 ng ml−1 rmIL-12 (R&D Systems) and 10 μg ml−1 anti-IL-4 (11B11; Biolegend). The TH17 cell differentiation conditions included 20 ng ml−1 rmIL-6 (R&D Systems), 3 ng ml−1 rmTGF-β1 (R&D Systems), 10 μg ml−1 anti-IL-4 (11B11; Biolegend) and 10 μg ml−1 anti-IFN-γ (XMG1.2; eBioscience). The iTreg-cell differentiation conditions included 5 ng ml−1 rmTGF-β1 (R&D Systems) and 10 ng ml−1 rmIL-2 (R&D Systems).
4
MDSC-Mediated T Cell Proliferation Assay
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Freshly isolated mouse BM cells were treated with GM-CSF and sB7H3 as described above. The control cell was treated with GM-CSF only. Three days later, the CD11b+Gr1+ MDSCs were isolated/purified using the EasySep™ Mouse Myeloid-Derived Suppressor Cell Isolation kit (Stemcell Technologies) according to the manufacturer’s instructions. Splenocytes isolated from mouse spleen were pre-labeled with 5 µM of proliferation dye carboxyfluorescein succinimidyl ester (CFSE) (BD Biosciences) and then co-cultured with GM-CSF/sB7H3-activated MDSC (1:1 ratio) in the presence of CD3/28 Dynabeads (Life Technology, Thermo Fisher Scientific) and 50 U/ml of rmIL2 (R&D Systems) for 3 days. The cells were collected and stained with CD4-BV421 (BioLegend, Cat# 100437, clone GK1.5) and CD8-APC (BioLegend, Cat# 100711, clone 53-6.7). The intensity of incorporated CFSE in CD4+ and CD8+ populations was analyzed by flow cytometry as described above.
5
MDSC Suppression of T-cell Proliferation
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Freshly isolated CD4+ splenocytes were stained with carboxyfluorescein-succinimidyl ester (CFSE, Invitrogen, Carlsbad, USA) according to the manufacturer´s instructions. Cells were suspended in RPMI 1640 media containing 1% P/S and 10% FCS. CFSE-labelled CD4+ T-cells (2x105) suspended in 100µl media were stimulated with 2x105 mouse T-Activator CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, USA) and 50 ng recombinant murine Interleukin-2 (rmIL-2, R&D Systems, Minneapolis, USA) under addition of ß Mercaptoethanol (Merck, Darmstadt, Germany) at a concentration of 50mM. MDSC isolated from spleens of pregnant WT and Qa-2- animals at E10.5 also suspended in RPMI 1640 containing 1% P/S and 10% FCS were added in different ratios (1:2, 1:4 and 1:8). After 3 days of culture, CD4+ T-cell proliferation was determined by flow cytometry using the CFSE dye dilution. Proliferation index, defined as the ratio of CD4+ T-cell proliferation after addition of MDSC and CD4+ T-cell proliferation without MDSC, was determined. CD4+ T-cell proliferation without MDSC was set to a fixed value of 1.
6
Isolation and Activation of Murine CD4+ T Cells
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Naive CD4+ CD62L+ T cells were isolated by using MicroBeads (Miltenyi Biotech, catalogue number: 130-104-453) or Dyna and Detacha Beads (Invitrogen, catalogue number: 11445D and catalogue number: 12406D, respectively) from spleens of 8- to 12-week-old C57BL/6 mice or of Bcl-3TOE and control littermates, and activated with plate-bound anti-CD3 (using first anti-hamster, Novartis, catalogue number: 55397 then anti-CD3 in solution: clone: 2C11H: 0.1 μg ml−1) and soluble anti-CD28 (clone: 37 N: 1 μg ml−1). TH0 and Treg cultures were additionally supplemented with blocking antibodies anti-IL-4 (clone: 11B11, 10 μg ml−1) and anti-IFN-γ (clone: Xmg-121, 10 μg ml−1). All antibodies were obtained in collaboration with and from Elisabeth Kremmer (Helmholtz Center Munich). For Treg differentiation additionally the following cytokines were added: rmIL-2 and rmTGF-β (both: R&D Systems, 5 ng ml−1). For expansion of Treg cells, cells were cultured in RPMI and 2,000 units Proleukin S (MP Biomedicals, catalogue number: 02238131). For experiments, only samples were used that achieved between 55–85% Foxp3 positive cells (Staining Kit, BD Bioscience: catalogue number: 00552300).
7
Splenocyte Proliferation and GRAIL Expression
8
Hepatic ILC2 Activation and CD4+ T Cell Interactions
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FACS-isolated hepatic ILC2 (1 × 104) were cultured in the presence of rmIL-33 (10 ng/ml), rmIL-25 (10 ng/ml), rmIL-1β (10–200 ng/ml; all BioLegend), rmIFNγ (10 ng/ml) and rmIL-12 (10–200 ng/ml; both R&D Systems, Wiesbaden, Germany) for 4 days. For ILC2 maintenance, all cultures were done in the presence of rmIL-2 (10 U/ml) and rmIL-7 (10 ng/ml; both R&D Systems). For CD4+ T-cell activation, hepatic ILC2 (2 × 104) were co-cultured with FACS-isolated, OVA-specific CD4+ T cells (1 × 105) or OVA-specific Th1 cells (1 × 105) in the presence of OVA323-339 peptide (5 µg/ml) for 4 days. For blocking IL-2 or IFNγ, co-cultures were done in the presence of an anti-IL-2 (JES-1A12; 10 µg/ml; BD Pharmingen) and anti-IFNγ (R4-6A2; 10 µg/ml; BioXCell, West Lebanon, NH) antibody, respectively.
9
Isolation and Activation of CD8+ T Cells
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CD8+ cells were enriched from spleen cell preparations via positive selection using anti-CD8a (Ly-2) microbeads (Miltenyi Biotech) and then CD45.2+CD3+CD8+ T cells were sorted by flow cytometry. 5x104 cells were seeded in wells of a 96-well plate with 100μL RPMI 1640 medium supplemented with 1mM sodium pyruvate, 10mM HEPES, 50μM mercaptoethanol, and 10% FCS with 40ng/mL rmIL-15 (Peprotech). Alternatively, wells were coated with anti-CD3 (5μg/mL, clone 145-2C11) and anti-CD28 antibodies (1μg/mL, clone 37.51) at 4°C overnight and then cells were added with 20ng/mL rmIL-2 (R&D systems) for 2 or 3d.
10
Naive CD4+ T Cell Isolation and Skewing
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CD4+ T cells were enriched from mice lymph nodes and spleen with anti-CD4 microbeads by using an AutoMACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer’s protocol. Thereafter, naive CD4+CD62LhighCD44lowT cells were purified using a FACSAria III cell sorter (BD Biosciences). The sort-purified naive CD4 T cells were TCR-activated with plate-bound anti-CD3ε (4 μg/mL) and anti-CD28 (2 μg/mL) (BD Biosciences) in complete IMDM (supplemented with 10% FBS, L-glutamine, penicillin-streptomycin and β–mercaptoethanol). Skewing conditions were as follows: 1 ng/mL rhTGF-β1 (eBioscience) plus 25 ng/mL rmIL-6 (R&D Systems) for cTH17; 25 ng/mL rmIL-6, 20 ng/mL rm-IL-1β and 30 ng/mL rmIL-23 (all from R&D Systems) for pTH17; 1 ng/mL rhTGF-β1 for iTreg; 20 ng/mL rmIL-12 and 20 ng/mL rmIL-2 (both from R&D Systems) for TH1. When indicated, CH223191 (30 μM; Tocris), DMXAA (3–30 μM; Invivogen), C-176 (1 μM; Sigma-Aldrich), c-di-AM(PS)2 (Rp,Rp) (15 μM; Invivogen), c-di-AMP or c-di-GMP (both 30–100 μM; Invivogen) were used.
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