For murine Th17 polarized culture, purified naive T cells were stimulated with plate-bound goat anti-hamster antibodies, soluble anti-CD3 Ab (0.25 μg/ml,145–2C11; Biolegend), anti-CD28 Ab (0.5 μg/ml, 37.51; Biolegend), IL-6 (10 ng/ml; R&D Systems), TGF-β1 (0.3 ng/ml; R&D Systems), anti-IL-4 Ab (10 μg/ml, C17.8; Biolegend), and anti-IFNγ Ab (10 μg/ml; XMG1.2; Biolegend). For the human Th17-polarized cultures, isolated naive CD4 T cells were stimulated with plate-bound anti-CD3 Ab (1 μg/ml, OKT-3; BioXCell), anti-CD28 Ab (1 μg/ml, CD28.2; Biolegend), IL-6 (50 ng/ml; # NM_000600; Biolegend), TGF-β1 (10 ng/ml, # NM_003236; Biolegend), IL-1β (10 ng/ml, # NM_000576; Biolegend), IL-23 (50 ng/ml, # NP_057668 and # NP_002178.2; Biolegend), anti-IL-4 Ab (10 μg/ml, MP4–25D2; BioXCell), and anti-IFNγ Ab (5 μg/ml; B27; BioXCell) (14 ). For the pharmacological Gls1 inhibition experiments, BPTES were diluted in 0.5 % DMSO and 10 mM BPTES or 0.5 % DMSO were administrated in the culture.
Mp4 25d2
Manufactured by BioXCell
The MP4-25D2 is a laboratory instrument designed for high-throughput microplate processing. It features 4 independent heating blocks that can accommodate up to 25 microplates simultaneously. The device is capable of maintaining precise temperature control within the range of ambient temperature to 100°C. This product is suitable for a variety of applications that require temperature-controlled microplate handling.
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Lab products found in correlation
Anti cd28 ab, by BioLegend (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Tgf β1, by BioLegend (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Mouse il 6, by Thermo Fisher Scientific (1 mentions)
Human il 6, by R&D Systems (1 mentions)
Human il 1β, by R&D Systems (1 mentions)
Human il 23, by R&D Systems (1 mentions)
Human tgf β, by R&D Systems (1 mentions)
2 protocols using mp4 25d2
1
Murine and Human Th17 Cell Polarization
2
Th17 Cell Differentiation Modulation
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Purified naive CD4+ T cells (2 to 4 ×105 per well) were cultured under Th17 differentiation conditions in 96-well round-bottom plates. Cells collected from mice were cultured in medium containing 5 μg/mL anti-mouse CD3 mAb (17A2, BioXcell), 1 μg/mL anti-mouse CD28 mAb (37.51.1, eBioscience), 50 μM 2-mercaptoethanol (S6250, Sigma), 5 ng/mL human TGF-β (R&D), 20 ng/mL mouse IL-6 (Peprotech), 5 μg/mL anti-mouse IFN-γ mAb (XMG1.2, BioXcell), and 5 μg/mL anti-mouse IL-4 mAb (11B11, BioXcell) for 3 days. Cells collected from humans were cultured in medium containing 5 μg/mL anti-human CD3 mAb (OKT3, eBioscience), 1 μg/mL soluble anti-human CD28 mAb (CD28.2, eBioscience), 50 μM 2-mercaptoethanol (Sigma), 2.5 ng/mL human TGF-β (R&D), 20 ng/mL human IL-6 (R&D), 10 ng/mL human IL-23 (R&D), 10 ng/mL human IL-1β (R&D), 5 μg/mL anti-human IFN-γ mAb (285-IF-100, R&D), and 5 μg/mL anti-mouse IL-4 mAb (MP4-25D2, BioXcell) for 6–7 days.
To determine the role of MDSCs and polyamines in Th17 cell differentiation, MDSCs (at a 1:1 ratio to naive CD4+ T cells) were added in the presence or absence of ornithine (ORN, 100 nM, Sigma), difluoromethylornithine (DFMO, 20 nM, Tocris), putrescine (PUT, 2.5 μM, Sigma), spermidine (SPD, 2.5 μM, Sigma), or spermine (SPM, 2.5 μM, Sigma).
To determine the role of MDSCs and polyamines in Th17 cell differentiation, MDSCs (at a 1:1 ratio to naive CD4+ T cells) were added in the presence or absence of ornithine (ORN, 100 nM, Sigma), difluoromethylornithine (DFMO, 20 nM, Tocris), putrescine (PUT, 2.5 μM, Sigma), spermidine (SPD, 2.5 μM, Sigma), or spermine (SPM, 2.5 μM, Sigma).
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