Naïve CD4 T cells were purified from splenocytes of WT mice using Miltenyi naïve CD4 isolation kit. For Th1 induction in Figure 1A , naïve CD4 T cells were cultured on 24-well plates coated with 1 μg/ml of αCD3/CD28 (Biolegend) plus IL-12 (0.5 ng/ml) and AS1842856 (or DMSO) for 72h. For iTreg induction in Figure 5 , αCD3/CD28 (1 μg/ml) and recombinant mouse PD-L1-Ig chimera or human IgG1-Ig (10 μg/ml) (Biolegend) were used to coat plates. Naïve CD4 T cells were cultured on coated plates plus TGFβ (4 μg/ml) and AS1842856 (or DMSO) for 72h. For the expansion of human Th1 cells in Figure 4 C –D , PBMCs from treatment-naïve MS patients were plated in flasks for 2–4 hrs to remove adherent cells. The suspension cells were then collected and activated with plate-bound αCD3/CD28 plus IL-12 (0.5 ng/ml) and AS1842856 (0.1 μM) or DMSO for 3 days.
αcd3 cd28
Manufactured by BioLegend
The αCD3/CD28 is a laboratory equipment product used for T-cell activation and expansion. It consists of antibodies targeted against the CD3 and CD28 receptors on the surface of T cells. This combination of antibodies provides the necessary signals to activate and proliferate T cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using αcd3 cd28
1
Induction of Th1 and iTreg Cells from Murine Naive CD4+ T Cells
2
Induction and Expansion of Th1 and iTreg Cells
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Naïve CD4 T cells were purified from splenocytes of WT mice using Miltenyi naïve CD4 isolation kit. For Th1 induction in Figure 1A, naïve CD4 T cells were cultured on 24-well plates coated with 1 g/ml of αCD3/CD28 (Biolegend) plus IL-12 (0.5 ng/ml) and AS1842856 (or DMSO) for 72h.
For iTreg induction in Figure 5, αCD3/CD28 (1 g/ml) and recombinant mouse PD-L1-Ig chimera or human IgG1-Ig (10 μg/ml) (Biolegend) were used to coat plates. Naïve CD4 T cells were cultured on coated plates plus TGFβ (4 µg/ml) and AS1842856 (or DMSO) for 72h. For the expansion of human Th1 cells in Figure 4 C-D, PBMCs from treatment-naïve MS patients were plated in flasks for 2-4 hrs to remove adherent cells. The suspension cells were then collected and activated with plate-bound αCD3/CD28 plus IL-12 (0.5 ng/ml) and AS1842856 (0.1 µM) or DMSO for 3 days.
For iTreg induction in Figure 5, αCD3/CD28 (1 g/ml) and recombinant mouse PD-L1-Ig chimera or human IgG1-Ig (10 μg/ml) (Biolegend) were used to coat plates. Naïve CD4 T cells were cultured on coated plates plus TGFβ (4 µg/ml) and AS1842856 (or DMSO) for 72h. For the expansion of human Th1 cells in Figure 4 C-D, PBMCs from treatment-naïve MS patients were plated in flasks for 2-4 hrs to remove adherent cells. The suspension cells were then collected and activated with plate-bound αCD3/CD28 plus IL-12 (0.5 ng/ml) and AS1842856 (0.1 µM) or DMSO for 3 days.
3
Evaluating Immune Cell Activation with CBD
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Spleen cells were obtained from naïve unmanipulated male C57BL/6 mice (6–10 weeks old, 18–22 g) and suspended in HBSS buffer (Beyotime Biotechnology Co., Ltd, Nantong, China) at 1 × 107 cells/mL. CFDA SE (5 mM) was added at a final concentration of 2.5 μM followed by a 5 min incubation at 37 °C. Then the cells were washed with complete RPMI-1640 medium and suspended in complete RPMI-1640 medium. The cells were implanted at 2 × 106 cell/well in a 24-well plate. αCD3/CD28 (Biolegend) were added at 500 ng/mL in each well. To these cultures, we added 50 μL serum obtained from experimental mice, along with various concentrations of CBD. The final volume of each well in 24-well plates was 1 mL. The cells were cultured for 72 h. Then the cells were washed with PBS, incubated with anti-CD8α and anti-CD4 antibody (Supplementary Table 2 ) for 45 min at 4 °C. After that, the cells were washed with PBS and the CFSE dilution was determined using flow cytometry (Beckman, coulter) by gating on CD8+ or CD4+ cells.
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