Spleens from naïve C57Bl/6 mice were digested for 30 min in RPMI media containing DNaseI (10 mg/ml) and Liberase TL (1.67 Wünsch Units/ml). T cells were isolated by MACS using the Pan T cell isolation kit (Miltenyi). Cells were fluorescently stained with anti-CD4-FITC (RM4-5, eBioscience), anti-C25-PECy5 (PC61.5, eBioscience), anti-CD44-PE (IM7, BioLegend) and anti-CD62L-APC (MEL-14, eBioscience) to isolate naïve T cells with a cell sorter. Naïve T cells were cultured on 96 well plates together with plate-bound anti-CD3 (2 µg/ml, 145-2C11, BD Pharmingen) and anti-CD28 (2 µg/ml, 37.51, BD Pharmingen) and rhTGF-β1 (1 ng/ml) for Treg cell differentiation. The cells were additionally treated with solvent (PBS), LA (250µM) or LA+PA (PA 150µM) for 4 days and analyzed for the frequency of CD25+FoxP3+ cells in CD4+ viable lymphocytes with flow cytometry. In some experiments, cells were additionally treated with an IL-10 receptor blocker (1µg/ml, Biolegend).
Anti cd62l apc mel 14
Manufactured by Thermo Fisher Scientific
Anti-CD62L-APC (MEL-14) is a fluorescent-labeled antibody that binds to the CD62L (L-selectin) cell surface marker. CD62L is involved in the homing of lymphocytes to lymphoid tissues. This antibody can be used to detect and quantify CD62L-expressing cells by flow cytometry.
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Lab products found in correlation
Anti cd4 fitc rm4 5, by Thermo Fisher Scientific (3 mentions)
Anti cd44 pe im7, by BioLegend (3 mentions)
Anti cd28, by BD (2 mentions)
Anti cd25 pe cy5 pc61, by Thermo Fisher Scientific (2 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (2 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
3 protocols using anti cd62l apc mel 14
1
Lipid Metabolism Regulates Regulatory T Cell Differentiation
2
Investigating TH17 Cell Differentiation Under ILA and Osmotic Stress
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Splenic T cells were isolated by magnetic activated cell sorting using the “Pan T cell isolation kit II” (Miltenyi Biotec) according to the manufacturer’s instructions. Isolated T cells were collected and re-suspended in MACS buffer at 3·107 cells/ml. For APC free differentiation, cells were fluorescently stained for 30 min in an antibody cocktail containing anti-CD4-FITC (RM4-5, eBioscience), anti-CD44-PE (IM7, BioLegend), anti-CD62L-APC (MEL-14, eBioscience) and anti-CD25-PE-Cy5 (PC61.5, eBioscience) and subsequently purified by fluorescence activated cell sorting on MoFlo (Beckman-Coulter). Sorted naive T cells (CD4+CD62L+CD44lowCD25neg) were stimulated by plate-bound anti-CD3 (2 μg/ml, 145-2C11, BD Pharmingen) and anti-CD28 (2 μg/ml, 37.51, BD Pharmingen) in the presence of IL-6 (40 ng/ml) and rhTGF-β1 (2 ng/ml). To determine the influence of indole-3 lactic acid on TH17 cell differentiation, cells were cultured with vehicle (0.1% Ethanol) or 10-500 μM indole-3-lactic acid (ILA) for 96h under isotonic or hypertonic (+40 mM NaCl) conditions.
3
Investigating TH17 Cell Differentiation Under ILA and Osmotic Stress
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