The spleens were extracted from Balb/c mice and naive CD4+ T cells were purified by using a magnetic cell sorting system (MACS® separation, Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were cultured in RPMI 1640 culture media with 10% heat-inactivated FBS (Cellgro, Herndon, VA, USA), 100 U/mL penicillin (Cellgro), 0.1 mg/mL streptomycin (Cellgro), 2 mM L-glutamine (Cellgro), and 0.05 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, USA) at 37°C in a 5% CO2-humidified incubator. Plate-bound anti-CD3 antibody (1 μg/mL, ebioscience) and anti-CD28 antibody (1 μg/mL, ebioscience) were used to stimulate T cells. Recombinant mouse IL-6 (25 ng/mL, BD, San Jose, CA, USA) and TGF-β (2.5 ng/mL, BD) were used for Th17 differentiation, and IL-2 (10 ng/mL, BD), IL-12 (5 ng/mL, Biosource, Camerillo, CA, USA), and anti-IL-4 (5 μg/mL) were added to induce differentiation into Th1 cells.
Plate bound anti cd3 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Plate-bound anti-CD3 antibody is a laboratory equipment product used for cell culture applications. It provides a means to activate T cells in vitro by cross-linking the CD3 complex on the T cell surface.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Recombinant mouse il 6, by BD (1 mentions)
Macs separation, by Miltenyi Biotec (1 mentions)
Anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Corning (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Tgf β, by BD (1 mentions)
Ne per extraction kit, by Thermo Fisher Scientific (1 mentions)
Soluble anti cd28, by Thermo Fisher Scientific (1 mentions)
Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by BioLegend (1 mentions)
Celltrace violet reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using plate bound anti cd3 antibody
1
Isolation and Differentiation of Murine Th17 and Th1 Cells
2
CD4+ T Cell Activation Signaling
3
Assessing CD4+ T Cell Proliferation in Murine B Cell Co-cultures
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Naïve CD4+ T cells were isolated from murine spleens by using a Naïve CD4+ T Cell Isolation Kit (Miltenyi Biotec) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) from a CellTrace™ CFSE Cell Proliferation Kit (Life Technologies). CFSE-labeled naïve CD4+ T cells at a concentration of 1~2 × 106 cells/ml were cultured alone or co-cultured with isolated B10 cells (1 × 106 cells/ml) from Ctrl, Cko, Ctrl × Il-10 KO, or Cko × Il-10 KO mice with plate-bound anti-CD3 antibody (5 μg/ml, Invitrogen) and soluble anti-CD28 antibody (2 μg/ml, Invitrogen). After 3 days, the cells were collected for an assessment of the CFSE dilution in the CD4+ T cells conducted by flow cytometry.
4
Stimulating CD4+ T Cell Activation
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Healthy control (HC) CD4+T cells were mixed with autologous or PBC CD19+CD24hiCD38hi B cells at a ratio of 5 : 1 in culture medium containing RPMI 1640 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). The cells were stimulated with 1 μg/mL plate-bound anti-CD3 antibody (Invitrogen, Carlsbad, CA) for 6 days, and cells were stimulated with 50 ng/mL PMA, 1 μg/mL ionomycin, and 1 μg/mL Brefeldin A (BioLegend, San Diego, CA, USA) for the last 6 h of culture to test for the production of intracellular IFN-r in CD4+T cells.
5
In vitro Expansion and Activation of T Cell Blasts
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Expansion of T cell blasts was obtained by incubating PBMCs for 72 h with anti CD3/CD28 beads in complete Panserin 401 medium. After 3 days, dead cells were removed by Ficoll-Plaque density gradient, and T-cell blasts were expanded in complete Panserin supplemented with IL-2 (100 IU/ml). Day 10 T-cell blasts were starved of IL-2 for 72 h, washed, incubated with CellTrace violet reagent (Invitrogen) for 8 min at 37 °C in the dark, and washed twice more. A total of 2 ×105 cells were seeded into 96-well plates and subjected to different stimuli (plate-bound anti-CD3 antibody (Invitrogen), soluble anti-CD3/CD28 beads (eBioscience), IL-2 at the concentration indicated in the figures) in the presence of absence of ruxolitinib (500 nM). The cells were cultured for 4 days, washed with PBS, and stained with anti-CD3, CD4, CD8, CD25, and CD69 antibodies prior to flow cytometry measurement.
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