T cells were purified from single-cell suspensions of spleen and lymph nodes from male and female mice aged 6–12 weeks by negative selection with biotinylated antibodies recognizing CD8 or CD4, CD19, B220, CD11b, CD11c, DX5, Ter119, and CD24 (UCSF Monoclonal Antibody Core) and magnetic anti-Biotin beads (MACSi Beads, Miltenyi Biotec). For IL-2 stimulation, purified T cells were pre-activated on 6-well plates coated with antibodies specific for CD3 (2C11) and CD28 (37.51) for 72 hours, removed, and cultured with rhIL-2 (100 u/mL, Roche) for 36 hours, and then cultured without rhIL-2 for the 36 hours prior to all experiments.
Rhil 2
Manufactured by Roche
Sourced in Germany, United States
RhIL-2 is a recombinant human interleukin-2 product. Interleukin-2 is a cytokine that plays a crucial role in the immune system, promoting the growth and differentiation of various immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Macsibeads, by Miltenyi Biotec (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Anti cd28 clone 37, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Rhtgf β, by R&D Systems (2 mentions)
Anti cd3 clone 145 2c11, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Negative magnetic selection, by Miltenyi Biotec (2 mentions)
Mem non essential amino acid, by Merck Group (2 mentions)
Ficoll paque plus, by Cytiva (2 mentions)
Rhil 12, by R&D Systems (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd3e, by BD (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
35 protocols using rhil 2
1
Purification and Activation of Mouse T Cells
2
Isolation and Activation of Murine T Cells
3
Isolation and Activation of Murine T Cells
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T cells were purified from single-cell suspensions of spleen and lymph
nodes from male and female mice aged 6–12 weeks by negative selection
with biotinylated antibodies (CD8, CD19, B220, CD11b, CD11c, DX5, Ter119 and
CD24, UCSF Monoclonal Antibody Core) and magnetic anti-Biotin beads (MACSi
Beads, Miltenyi Biotec). For IL-2 stimulation, purified T cells were
pre-activated on 96-well plates coated with anti-CD3 (2C11) and anti-CD28
(37.51) for 72 hours, removed and cultured with rhIL-2 (100 u/mL, Roche) for 36
hours, and then cultured without rhIL-2 for the 36 hours prior to all
experiments.
nodes from male and female mice aged 6–12 weeks by negative selection
with biotinylated antibodies (CD8, CD19, B220, CD11b, CD11c, DX5, Ter119 and
CD24, UCSF Monoclonal Antibody Core) and magnetic anti-Biotin beads (MACSi
Beads, Miltenyi Biotec). For IL-2 stimulation, purified T cells were
pre-activated on 96-well plates coated with anti-CD3 (2C11) and anti-CD28
(37.51) for 72 hours, removed and cultured with rhIL-2 (100 u/mL, Roche) for 36
hours, and then cultured without rhIL-2 for the 36 hours prior to all
experiments.
4
Investigating T Cell Activation Pathways
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P14, Pmel, and OT-1 CD8+ T cells (1 × 106 cells per sample) were isolated and incubated with APNs (10 μg/ml) in fluorescence-activated cell sorting (FACS) buffer (1× Dulbecco’s PBS + 2% FBS + 1 mM EDTA + 25 mM HEPES) for 30 min at 37°C. Cells were washed three times with 1 ml of FACS buffer before analysis on a BD Accuri C6. For validation of in vitro transfection, P14 CD8+ T cells were activated for 24 hours as described above and resuspended in T cell media + rhIL-2 (30 U/ml; Roche) at 2 × 106 cell/ml. Cells (5 × 105) were coincubated with GP33/Db APN containing eGFP mRNA (1 μg) in 24-well plates at 37°C. After 4 hours, 700 μl of T cell media + rhIL-2 (30 U/ml; Roche) was added to each well. After an additional 48-hour incubation, cells were washed three times and stained against αCD8 monoclonal antibody (mAb; clone 53-6.7, BioLegend; table S2) at 4°C for 30 min. Cells underwent another two washes with FACS buffer before analysis on BD Accuri C6.
5
Isolation and Expansion of Human T Cells
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PBMCs were isolated by the Ficoll-Hypaque density gradient centrifugation of venous blood samples and cryopreserved at −150°C until use. Thawed PBMCs were allowed to rest temporarily during experiments in RPMI-1640 medium with GlutaMAX supplemented with 10% FBS (lymphocyte medium). The HEK293T cell line was purchased from the ATCC and cultured in DMEM supplemented with 10% FBS. The HuT78 T-lymphoma and Raji B-lymphoma cell lines were purchased from the ATCC and cultured in lymphocyte medium. T-blasts were generated by culturing PBMCs in ImmunoCult XF T Cell Expansion Medium (STEMCELL Technologies) supplemented with recombinant human interleukin 2 (rhIL-2; Cat: 11147528001; Roche) at a final concentration of 10 ng/ml and ImmunoCult Human CD3/CD28/CD2 T Cell Activator (1:100; STEMCELL Technologies), as previously described (Ogishi et al., 2023 (link)).
6
Detection of ANA in Mouse and Human Lymphocytes
7
Foxp3 Treg Cell Differentiation
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CD4+CD44loFoxp3− naive T cells from Foxp3-Tocky mice were isolated by cell sorting, and 105 cells were cultured on anti-CD3 (clone 1452C11; 2 µg/ml; eBioscience) and anti-CD28 (clone 37.51; 10 µg/ml; eBioscience)–coated 96-well plates (Corning) in the presence of 100 U/ml rhIL-2 (Roche) and 2 ng/ml rhTGFβ (R&D) for 0–48 h in a final volume of 200 µl RPMI 1640 (Sigma-Aldrich) containing 10% FCS and penicillin/streptomycin (Thermo Fisher Scientific).
Mature Foxp3+ Treg cells from Foxp3-Tocky mice were isolated by cell sorting, and 105 cells were cultured on anti-CD3 (clone 145.2C11; 2 µg/ml) and anti-CD28 (clone 37.51; 10 µg/ml) –coated 96-well plates in the presence of 100 U/ml rhIL-2 for 0–48 h in a final volume of 200 µl RPMI 1640 containing 10% FCS and penicillin/streptomycin.
Mature Foxp3+ Treg cells from Foxp3-Tocky mice were isolated by cell sorting, and 105 cells were cultured on anti-CD3 (clone 145.2C11; 2 µg/ml) and anti-CD28 (clone 37.51; 10 µg/ml) –coated 96-well plates in the presence of 100 U/ml rhIL-2 for 0–48 h in a final volume of 200 µl RPMI 1640 containing 10% FCS and penicillin/streptomycin.
8
Assessment of IL-10 and IFN-γ expression in Cor93 T cells
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To test the expression and production of IL-10 and IFN-γ by Cor93 TE, cells were incubated in complete RPMI 1640 media with 2 µg/ml of rhIL-2 (Roche) at 5×106 cells/ml for 4 h at 37 °C in the presence or absence of the Cor93 peptide. To assess the effect of IL-10 on Ag-induced apoptosis, Cor93 CD8 TE (107 cells/ml) were incubated for 1 h at 37 °C with 18 µg/ml of anti-IL-10R Ab (BioXCell), 400 ng/ml of recombinant mIL-10 (Biolegend) or left untreated prior to the addition of Cor93 peptide (1 mg/ml) and human IL-2 (1 mg/ml). For the assessment of cell viability, cells were harvested 8 h later and stained with Annexin V and 7AAD, as described above. For the assessment of CD25 expression, cells were harvested 24 h after peptide stimulation. For STAT5 phosphorylation, Cor93 CD8 TE were cultured overnight in serum-free RPMI complete medium (Gibco, Life Technologies), prior to treating them as described above. Cells were harvested 15 min after peptide stimulation.
9
Induction of Regulatory T Cells from Naïve T Cells
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Naïve T cells from Foxp3‐Tocky mice were isolated by negative selection using immunomagnetic selection (StemCell Technologies) and 2 × 105 cells cultured on anti‐CD3 (clone 1452C11, 2 μg/ml) and anti‐CD28 (clone 37.51, 10 μg/ml; both eBioscience)‐coated 96‐well plates (Corning) in the presence of 500 U/ml rhIL‐2 (Roche) and 5 ng/ml rhTGFβ (R&D) for 48 h in a final volume of 200 μl RPMI1640 (Sigma) containing 10% FCS and penicillin/streptomycin (Life Technologies). Cells were harvested then incubated with 100 μg/ml cycloheximide (Sigma) for the indicated time points before analysis by flow cytometry. For determination of the red half‐life, Blue−Red+ CD4+ T cells from Nr4a3‐Tocky mice were isolated by cell sorting and then cultured without TCR signals under the presence of rhIL‐2 for the indicated number of hours before analysis by flow cytometry. Importantly, IL‐2 signals do not induce Timer expression in Nr4a3‐Tocky T cells (Bending et al, 2018 ).
10
Isolation and Expansion of Primary Human NK Cells
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Primary human NK cells were isolated from the peripheral blood of healthy donors from the National Blood Service under ethics licence REC 05/Q0401/108 (University of Manchester, UK). Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained by separation on a Ficoll-Paque gradient. NK cells were acquired from PBMCs via negative selection using an NK cell isolation kit (Miltenyi Biotec). NK cells were maintained in Dulbecco's Modified Eagle Medium, 30% F12 Ham, 10% human serum, 1% non-essential amino acids, 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM L-glutamine, 50 U/ml penicillin streptomycin and 50 µM 2-mercaptoethanol (Gibco). NK cells were stimulated with rhIL-2 (200 U/ml; Roche) and rested for 6 days before experiments. Cells were kept at 37°C in a 5% CO2 atmosphere.
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