We assessed T-cell proliferation in patients’ peripheral blood samples from the GV1001 group (n = 75). We thawed the peripheral blood mononuclear cells (PBMCs) at 1 × 106 cells in RPMI 1640 (GIBCO, USA) with 10% human albumin serum (Sigma, USA) and penicillin-streptomycin (GIBCO, USA) with 2 µM CSFE (5, 6-carboxyfluorescein diacetate succinimidyl ester; BD Biosciences, USA). PBMCs were seeded in 96-well plates (BD Biosciences, USA) at 5 × 105 cells per well. The cells were cultured with coated 1 µg/ml of Anti-CD3 (BD Biosciences, USA), 1 µg/ml of anti-CD28 (BD Biosciences, USA), and further incubated with 20 µg/ml of GV1001 for stimulation in 4 days at 37 °C. After 4 days, the cells were harvested, stained with fluorochrome-labelled Anti-CD3 (BD Biosciences, USA), and analysed using flow cytometry (FACSVerse, BD Biosciences). We defined a positive proliferative response to GV1001 as a stimulation index >2 with a significant difference in CFSE fluorescence compared between days 0 and 4. For subpopulation analysis, we stained 1 × 106 PBMC with Anti-CD3, anti-CD4, and anti-CD8 (BD Biosciences, USA) and analysed them using flow cytometry (FACSVerse, BD Biosciences).
Anti cd3
Manufactured by BD
Sourced in United States, Germany, United Kingdom, China, Belgium, Japan, Denmark, Australia
Anti-CD3 is a monoclonal antibody that binds to the CD3 complex on the surface of T cells. The CD3 complex is essential for T cell activation and signal transduction. Anti-CD3 can be used in various applications, such as flow cytometry and cell culture experiments, to identify and study T cell populations.
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Lab products found in correlation
Anti cd28, by BD (176 mentions)
Anti cd4, by BD (92 mentions)
Anti cd8, by BD (85 mentions)
Anti cd11b, by BD (37 mentions)
Soluble anti cd28, by BD (36 mentions)
Anti cd19, by BD (35 mentions)
Anti cd45, by BD (35 mentions)
Anti ifn γ, by BD (30 mentions)
Anti cd11c, by BD (23 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (21 mentions)
Ionomycin, by Merck Group (19 mentions)
Anti cd56, by BD (18 mentions)
Anti b220, by BD (17 mentions)
Facscanto 2, by BD (16 mentions)
Anti il 4, by R&D Systems (16 mentions)
Interleukin 2 (il 2), by R&D Systems (16 mentions)
Anti cd28 antibody, by BD (16 mentions)
Facsdiva software, by BD (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Phorbol myristate acetate (pma), by Merck Group (13 mentions)
541 protocols using anti cd3
1
Evaluating T-cell Proliferation in GV1001 Immunotherapy
2
Naive CD4+ T Cell Isolation and Activation
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Naive CD4 1 T cells (CD4 1 CD62L hi CD44 2 ) were sorted from a single-cell suspension drawn from spleens with the Na€ ıve CD4 1 T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and purity levels were greater than 95%, as determined by using the BD FACSCelesta. Naive T cells were used for in vitro cultures in RPMI 1640 medium (Corning, Manassas, Va) supplemented with b-mercaptoethanol, 10% FBS, and 1% penicillin-streptomycin. For T-cell activation, proliferation, and viability detection, naive T cells were stimulated with or without anti-CD3 (precoated, 5 mg/mL) and anti-CD28 (1 mg/mL; BD Biosciences, San Jose, Calif) for 3 days. For T H 2 cell differentiation, naive CD4 1 T cells were differentiated under T H 2-skewed conditions: mIL-2 (10 ng/ mL), mIL-4 (50 ng/mL), and anti-IFN-g (10 mg/mL; Miltenyi Biotec) with anti-CD3 (precoated, 5 mg/ml) plus anti-CD28 (1 mg/mL; BD Bioscience) for 7 days. In pharmacologic inhibition experiments T cells were pretreated with mock stimulant or 2-deoxy-D-glucose (2-DG; 1 mmol/L; Santa Cruz Biotechnology, Santa Cruz, Calif), rapamycin (100 nmol/L; Sigma-Aldrich, St Louis, Mo), and 5-aza-29-deoxycytidine (1 mmol/L; MedChemExpress, Monmouth Junction, NJ) and then stimulated with anti-CD3 (precoated, 5 mg/mL) and anti-CD28 (1 mg/mL; BD Biosciences) or T H 2-skewed conditions.
3
T Cell Proliferation and Polarization
4
Isolation and Activation of CD4+ T-cells
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T-cells were obtained from lung, spleen, thymus, or tracheobronchial lymph nodes (tLN). Mouse naïve CD4+ T-cells (CD4+CD62LhiCD44−) were sorted by Naïve CD4+ T-cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Human CD4+ T-cells were sorted by CD4+ T-cell Isolation Kit (Miltenyi Biotec). The purity levels were above 95% detected by BD FACSCelestaTM. For in vitro experiments, mouse CD4+ T-cells were cultured in RPMI 1640 medium (Corning, Manassas, VA, USA) supplemented with 10% FBS, and 1% penicillin-streptomycin, and then stimulated by anti-CD3 (precoated, 5 μg/mL; BD Biosciences) and anti-CD28 (1 μg/mL; BD Biosciences) for 3 h. For TH2 cell differentiation, naïve CD4+ T-cells were cultured under the following conditions: mIL2 (10 ng/mL; BD Bioscience), mIL4 (50 ng/mL; BD Bioscience), and anti-IFN-γ (10 μg/mL; BD Bioscience) with anti-CD3 (precoated, 5 mg/mL) plus anti-CD28 (1 mg/mL; BD Bioscience) for 5–7 days. Human CD4+ T-cells were stimulated by PMA (50 ng/mL; Sigma) and ION (500 ng/mL; Sigma) for 3 h. In pharmacologic inhibition experiments, T cells were pretreated with 5-aza-2′-deoxycytidine (1 μg/mL; MedChemExpress, Monmouth Junction, NJ, USA) and then stimulated with anti-CD3 (precoated, 5 μg/mL; BD Biosciences) and anti-CD28 (1 μg/mL; BD Biosciences) for 3 h.
5
Cytokine-Induced CD4 T Cell Activation
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Sorted human memory CD4 T cells were cultured in 48-well plates (5×10 5 cells per well) containing platebound anti-CD3 (5 µg/ml, BD Bioscience) and soluble anti-CD28 (1 µg/ml, BD Bioscience) supplemented with 20 ng/ml IL-1β (Peprotech). For quantitative real-time PCR and ELISA, cells or supernatants were collected on days 1 and 2. Murine splenic CD4 T cells were enriched by a mojoSort™ mouse CD4 T cell isolation kit (Biolegend), cultured with plate-bound anti-CD3 (2 µg/ml, BD Bioscience) and soluble anti-CD28 (1 µg/ml, BD Bioscience), and stimulated with 20 ng/ml IL-1β (Peprotech) for 30 min to study the phosphorylation of signaling molecules.
6
CD4 T Cell Activation with Anti-CD3/CD28 and IL-1β
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Sorted human memory CD4 T cells were cultured in 48-well plates (5 × 105 cells per well) containing plate-bound anti-CD3 (5 μg/ml, BD Bioscience) and soluble anti-CD28 (1 μg/ml, BD Bioscience) supplemented with 20 ng/ml IL-1β (Peprotech). For quantitative real-time PCR and ELISA, cells or supernatants were collected on days 1 and 2. Murine splenic CD4 T cells were enriched by a mojoSort™ mouse CD4 T cell isolation kit (Biolegend), cultured with plate-bound anti-CD3 (2 μg/ml, BD Bioscience) and soluble anti-CD28 (1 μg/ml, BD Bioscience), and stimulated with 20 ng/ml IL-1β (Peprotech) for 30 min to study the phosphorylation of signaling molecules or 48 h for cytokine production by ELISA or flow cytometry.
7
Th1 and Th2 Cell Polarization
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CD4 T cells were isolated from mesenteric lymph nodes of naïve mice using flow cytometry (>99% purity). Sorted CD4 T cells were plated at 1 × 105 cells/well on plates coated with 10 μg ml−1 anti-CD3 (BD Bioscience) and 5 μg ml−1 anti-CD28 (BD Bioscience). Th1 polarization conditions: 5 ng ml−1 rIL-12 (BD Bioscience) and 50 μg ml−1 anti-IL-4 (homemade Clone: 11B11); Th2 polarization: 50 ng ml−1 rIL-4 (BD Bioscience) and 50 μg ml−1 anti-IFN-γ. Cells were cultured in a final volume of 100 μl for 72 hrs, then transferred to fresh round-bottom 96 well plates and resuspended in appropriate antibody cocktails with the addition of 20 U ml−1 IL-2 (BD Bioscience) and cultured for another 48 hrs. Finally, cells were plated at 2 × 105 cells/well and incubated in 96 well plates coated with 20 μg ml−1 anti-CD3 (BD Bioscience). Supernatants were harvested after 48 hrs restimulation and used for ELISA.
8
T Cell Stimulation and Culture Protocols
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Human T cells were stimulated with plate-bound anti-CD3 (1 µg/ml) and soluble anti-CD28 (1 µg/ml) (BD Bioscience) antibodies in RPMI 1640 medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM l -glutamine (all from Life Technologies Invitrogen), 10% normal human serum (NHS), and 10 U/ml IL-2 (Novartis) for the indicated amount of time.
Murine T cells were cultured without stimulation or stimulated with plate-bound anti-CD3 (2 µg/ml) and anti-CD28 (10 µg/ml) (BD Bioscience) antibodies in RPMI 1640 medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mMl -glutamine, 10% FCS (all from Life Technologies Invitrogen), and 10 ng/ml IL-2 (R&D Systems) for the indicated amount of time. Mouse TGFβ1 (2 ng/ml, R&D Systems) was added when indicated. For serum starvation, cells were incubated with serum-free RPMI 1640 medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM l -glutamine (Life Technologies Invitrogen) and 10 ng/ml IL-2 (R&D Systems) for 20 h.
Murine T cells were cultured without stimulation or stimulated with plate-bound anti-CD3 (2 µg/ml) and anti-CD28 (10 µg/ml) (BD Bioscience) antibodies in RPMI 1640 medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM
9
Th0 and Th17 Cell Differentiation
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Isolated PBMC cells (5×105) from healthy individuals were incubated under appropriate conditions for 48 hours. To induce Th0 differentiation, PBMCs (5×105) were incubated for 48 hours with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies (BD Biosciences, San Diego, CA, USA). To induce Th17 differentiation, PBMCs (5×105) were incubated for 48 hours with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies (BD Biosciences), IL-1β (20 ng/mL) (R&D Systems, Inc. Minneapolis, MN, USA), IL-6 (20 ng/mL) (R&D Systems), IL-23 (20 ng/mL) (R&D Systems), and IFN-γ–neutralizing antibody (2 μg/mL) (R&D Systems), and IL-4-neutralizing antibody (2 μg/mL) (R&D Systems).
10
Immunosuppressive effects of dexamethasone and glucagon on T cell function
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Isolated TCD4+ cells (2 × 104/well) were obtained from A/J mice and stimulated with immobilized anti-CD3 plus anti-CD-28 (0.5 µg/ml and 1 µg/mL, respectively; BD Biosciences Pharmingen). Cells were incubated with dexamethasone (1 μM) or glucagon (3 μM) for 72 h at 37 °C in 5% CO2. Proliferation was measured by BrdU incorporation using the Cell Proliferation ELISA, BrdU colorimetric kit (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. To evaluate the production of cytokines IL-2, IL-4, IL-10, and TNF-α, isolated TCD4+ cells (5 × 105 cells/well) were stimulated with immobilized anti-CD3 plus anti-CD-28 (1 and 2 µg/mL, respectively) and were detected from the supernatant of these cells using a CBA kit (BD Biosciences PharMingen).
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