Naive (CD4+CD25−CD62LhighCD44low) and memory-like (CD4+CD25−CD62LlowCD44high) T cells from the spleens and lymph nodes of 7–12-week-old mice were isolated using a FACS Aria cell sorter II (BD Biosciences, Franklin Lakes, NJ, USA). Purified naive and memory-like CD4+ T cells were stimulated with IL-1β (20 ng/mL, R&D Systems, Minneapolis, MN, USA), IL-23 (20 ng/mL, R&D Systems), IL-7 (10 ng/mL, Peprotech, Rocky Hill, NJ, USA), or plate-bound anti-CD3/CD28 (2 μg/mL, BD Biosciences). Cytokines and other reagents used in further cell culture experiments included TNF (20 ng/mL, Peprotech), IL-12 (10 ng/mL, Peprotech), IL-1RA (100 ng/mL, R&D Systems), Bay 11-7082 (1 μM, Calbiochem, San Diego, CA, USA), SB203580 (1 μM, Calbiochem), and CsA (50 ng/mL, Calbiochem). To generate OT-II memory-like TH17 cells in vitro, naive (CD4+Vα11+CD25−CD62LhighCD44low) T cells were sorted from either OT-II or IL-1R1-deficient (Il1r1−/−) OT-II mice. These CD4+ T cells were primed with anti-CD3/CD28 (4 μg/mL) in the presence of TGF-β (0.5 ng/mL, R&D Systems), IL-6 (30 ng/mL, BD Biosciences), and IL-23 (20 ng/mL, BD Biosciences). After a 4-day priming period, the OT-II TH17 cells were washed and further cultured in medium containing IL-7 (10 ng/mL, Peprotech) for 8–10 days.
Anti cd3 cd28
Manufactured by BD
Sourced in United States
Anti-CD3/CD28 is a laboratory reagent used to activate and expand T cells in vitro. It consists of antibodies that bind to the CD3 and CD28 surface receptors on T cells, providing the necessary signals to stimulate T cell activation and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Il 12, by Thermo Fisher Scientific (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by BD (2 mentions)
Il 23, by R&D Systems (2 mentions)
Tgf β, by R&D Systems (2 mentions)
Class b cpg oligodeoxynucleotides, by InvivoGen (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Concanavalin a (cona), by Merck Group (2 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Facs aria cell sorter 2, by BD (1 mentions)
Anti cd3 cd28, by R&D Systems (1 mentions)
Il 23, by BD (1 mentions)
Tumor necrosis factor (tnf), by Thermo Fisher Scientific (1 mentions)
Il 1ra, by R&D Systems (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Ack lysis buffer, by Lonza (1 mentions)
Paramagnetic beads, by STEMCELL (1 mentions)
Lsr 2, by BD (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
10 protocols using anti cd3 cd28
1
Generating Memory-like TH17 Cells
2
Murine Splenocyte Cytokine Profiling
3
Cytokine Profiling of Activated Lymphocytes
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Lymphocytes from DLN (mediastinal and hilar) and purified splenic CD4+ cells were cultured in RPMI1640 medium (5% FCS) and were stimulated with medium as control, OVA (50 µg/ml) for 5 days or anti-CD3/CD28 (BD Bioscience) (5 µg/ml) for 3 days. Supernatants were collected for cytokine measurement.
4
CD4+ T Cell Activation and Autophagy
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CD4 positive T (CD4+ T) cells were isolated from peripheral mononuclear cells using paramagnetic beads (Stemcell technologies) according to the manufacturer’s instructions. Cell purity was > 90%, which was confirmed by BD LSR II (BD Biosciences). CD4+ T cells were labeled using carboxyfluorescein succinimidyl ester (CFSE) (BioLegend) according to the manufacturer’s instructions, cultured in the presence of anti-CD3/CD28 (BD Biosciences) (each at final concentration of 2.5 μg/ml) for 2 days and then incubated with A549-atg5-wt or A549-atg5-null cells at a T-cell: A549 cell ratio of 1:5 for the next 2 days. The samples were analyzed by BD LSR II and the results represent the mean ± SD of technical triplicates, each with 30,000 counted cells.
5
Memory B cell function with CD8+ T cells
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CD19+ CD27+ memory B (MB) cells were isolated using the EasySep Human Memory B Cell Isolation Kit (STEMCELL Technologies, Canada) according to the manufacturer’s instructions. CD8+T cells were isolated using a CD8+ T Cell Isolation Kit (Miltenyi, Germany). MB cells (1×105 /mL) from SLE and Healthy donors were cultured alone or in the presence of autologous CD8+ T cells (4×105 /mL) at 1:4 ratio for 3 days at 37°C. Co-cultured cells were incubated in complete RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen) supplemented with 25 mM HEPES (Invitrogen), 2 mM L-glutamate (Invitrogen) and 1% non-essential amino acids (Life Technologies). During incubation, cells were stimulated with class B CpG oligodeoxynucleotides (InvivoGen) at 5 µg/mL, 1 µg/mL anti-CD3/CD28 (BD Biosciences, USA).
6
Regulatory T Cell Activation Assay
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In most experiments, CD4+CD25+ purified T cells were cultured at a concentration of 0.5 × 106 cells/ml per well in triplicate for each condition and resuspended in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Waltham, MA, USA), 2 mM L-glutamine, 0.1% gentamicin, and 50 mM β-Mercaptoethanol. Plates were incubated with plate-bound anti-CD3/CD28 (1 and 0.25 μg/ml, respectively) (BD Biosciences) and different amounts of rIL-2 (Peprotech, Rocky Hill, NJ, USA) as indicated for 72 h at 37°C, 5%CO2 incubator. In some experiments the proteasome inhibitor MG-132 (Sigma-Aldrich) was added at a concentration of 5 μM during the last 12 h of the culture.
7
Induction and Manipulation of CD4+ T Cell Subsets
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Naive (CD4+CD25−CD62LhighCD44low) T cells from the spleens and lymph nodes of 6- to 9-week-old mice were isolated using a Naive T Cell Isolation Kit II (Miltenyi Biotec). Purified naive CD4+ T cells were stimulated with plate-bound anti-CD3/CD28 (2 μg/mL; BD Biosciences). The cells were incubated in the presence of the following cytokines for 3 or 5 days: anti-IL-4 neutralizing antibody (5 μg/mL; BD Biosciences), IL-2 (50 U/mL; PeproTech), and IL-12 (2 ng/mL; PeproTech) for Th1; anti-IFN-γ neutralizing antibody (5 μg/mL; BD Biosciences), IL-4 (30 ng/mL; BD Biosciences), and IL-2 (50 U/mL; PeproTech) for Th2 cells; anti-IL-4/IFN-γ neutralizing antibody (5 μg/mL; BD Biosciences), TGF-β (1 ng/mL; R&D Systems), IL-6 (30 ng/mL; BD Biosciences), IL-1β (20 ng/mL; R&D Systems), and IL-23 (20 ng/mL; R&D Systems) for Th17; IL-2 (100 U/mL; PeproTech) and TGF-β (5 ng/mL; R&D Systems) for iTreg cells. Th2 cells were harvested at day 5 and were further reactivated by plate-bound anti-CD3/CD28 (2 μg/mL) for 24 h. Cells were incubated with dNP2-VEET (1 μM), dNP2-VIVIT (1 μM), and CsA (20 ng/mL; Calbiochem).
8
Resveratrol Modulates Immune Cell Activation
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Mice were euthanized 7 months after receiving the single pristane injection and spleens were removed for analysis. CD4+ T cells and CD19+ B cells from splenic mononuclear cells (SMC) were purified by positive selection (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin. Cells were cultured at 37°C in a 5% CO2 incubator for the indicated time periods. SMCs and CD4+ T cells were activated with ConA (Sigma-Aldrich, USA), anti-CD3 (BD Biosciences PharMingen, USA), or anti-CD3/CD28 (BD Pharmingen, USA) with or without resveratrol (0, 10, 20, 40, or 80 µM) for the indicated time points. CD19+ B cells were activated with lipopolysaccharide (LPS) with or without resveratrol (0, 10, 20, 40, or 80 µM).
9
PD-L1 Overexpression in Systemic Lupus Erythematosus
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Recombinant lentiviral overexpression of PD-L1 was a gift from Wuhan University. The viral supernatant and medium were mixed in a 1:1 ratio, and the total volume was 1 mL. Polybrene was added at a concentration of 8 µg/mL. MB cells (1×105 cells from patients with SLE) were resuspended in this mixture and inoculated into 24-well culture plates for 24 hours. The expression of PD-L1 on the surface of MB cells was detected by flow cytometry. Transfected MB cells (1×104 transfected MB cells) were cultured with CD8+T cells from healthy donors or patients with SLE at a ratio of 1:4 for 72 hours. Class B CpG oligodeoxynucleotides (InvivoGen) at 5 µg/mL, 1 µg/mL anti-CD3/CD28 (BD Biosciences, USA) were added to the culture as stimulants. IFN-γ expression in CD8+ T cells was measured using flow cytometry.
10
Splenocyte Proliferation Assay
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After 8 or 12 weeks, mice were euthanised and spleens were collected aseptically, weighed and single-cell suspensions were prepared as previously described (24) . Splenocytes were seeded into ninety-six-well round-bottom cell culture plates (2 × 10 5 /well) and incubated in the presence of T-cell mitogens: concanavalin A (Con A) (Sigma-Aldrich) at 0•5, 1•5 or 3 µg/ml; phytohaemagglutinin (PHA) (Difco Laboratories) at 2•5, 5 or 20 µg/ml; or platecoated anti-T-cell receptor antibodies (anti-CD3) at 1 or 5 µg/ml without or with soluble anti-CD28 (anti-CD3/CD28; BD Biosciences) at 2 µg/ml for 72 h. Cultures were pulsed with 0•5 µCi [ 3 H]-thymidine during the final 4 h of incubation. Cells were harvested onto glass fibre filter mats, and cell proliferation was quantified as the amount of [ 3 H]-thymidine incorporation into DNA by liquid scintillation counting in a 1205 Betaplate counter (Wallac). Data are expressed as counts per min.
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