Lymph nodes and spleens were pooled, and CD4+ T cells were enriched by depletion. Non-CD4 T cells were stained with anti-CD8a (53–6.7), B220 (RA3–6B2), CD11c (N418), CD11b (M1/70), and CD49b(DX5) biotinylated antibodies (Biolegend) followed by magnetic microbead selection (Miltenyi). Cells were sorted on FACSAria III or FACSAria Fusion instruments (BD). Th0 cultures were harvested after 12 hours and stained with fluorochrome-conjugated anti-CD4 and Zobmie Auqua Live/Dead viability dye and sorted as described above.
Magnetic microbead selection
Manufactured by Miltenyi Biotec
Sourced in Germany
Magnetic microbead selection is a lab equipment product that uses magnetic beads to isolate specific cells or molecules from a sample. It allows for the separation and purification of target analytes based on their binding to the magnetic beads. The core function of this product is to facilitate the efficient extraction and enrichment of desired components from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd49b dx5 biotinylated antibodies, by BioLegend (2 mentions)
Facsaria 3, by BD (2 mentions)
Facsaria fusion instrument, by BD (2 mentions)
Cd11b m1 70, by BioLegend (2 mentions)
Biotinylated antibodies, by BioLegend (2 mentions)
Anti cd3 145 2c11, by BioLegend (2 mentions)
Anti cd28 37, by BioLegend (2 mentions)
Human tgf β1, by BioLegend (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Recombinant mouse il 2, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Anti cd3 145 2c11, by Merck Group (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Click s media, by Merck Group (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using magnetic microbead selection
1
CD4+ T Cell Isolation and Enrichment
2
In Vitro Treg Cell Induction
3
Culturing Melanoma Cells and Neutrophils
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HCmel3 melanoma cells and bone marrow‐derived neutrophils (BMDNs) were cultured in complete RPMI 1640 medium containing 10% FCS (Biochrom, Cambourne, UK), 2 mm l ‐glutamine (Gibco, Dublin, Ireland), 10 mm non‐essential amino acids (Gibco, 1 mm HEPES), 20 µm 2‐mercaptoethanol (Sigma‐Aldrich, St. Louis, MO, USA), 100 IU mL–1 penicillin and 100 mg mL–1 streptomycin (Invitrogen, Waltham, Massachusetts, USA). All cells were incubated at 37°C, 5% CO2 and 21% O2, apart from hypoxia experiments when 2% O2 was used. For isolation of BMDNs, the bone marrow from Ifnar1‐competent and Ifnar1‐deficient mice was harvested from femurs and tibias under sterile conditions and suspended in PBS. Erythrocytes were removed by hypotonic lysis. BMDNs were sorted using magnetic microbead selection following the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). A negative selection against CD11c+, DX5+, CD4+, CD8+ and CD19+ cells was first performed, and the remaining cells were positively selected for Ly6G+ cell population. Cells were cultured in RPMI medium (1640; Gibco). The purity of MACS‐sorted cells was confirmed by flow cytometry.
4
CD4+ T Cell Isolation and Enrichment
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Lymph nodes and spleens were pooled, and CD4+ T cells were enriched by depletion. Non-CD4 T cells were stained with anti-CD8a (53–6.7), B220 (RA3–6B2), CD11c (N418), CD11b (M1/70), and CD49b(DX5) biotinylated antibodies (Biolegend) followed by magnetic microbead selection (Miltenyi). Cells were sorted on FACSAria III or FACSAria Fusion instruments (BD). Th0 cultures were harvested after 12 hours and stained with fluorochrome-conjugated anti-CD4 and Zobmie Auqua Live/Dead viability dye and sorted as described above.
5
In Vitro Treg Cell Induction
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Treg cell induction in vitro was performed by culturing 0.2 × 106 sorted T cells/well in 96 well plates pre-coated with anti-CD3 (145–2C11) (1 μg/mL) using Click’s media (Millipore-Sigma) containing 10% fetal bovine serum (FBS) (Gemini); penicillin-streptomycin, L-glutamine, HEPES, β-mercaptoethanol, sodium pyruvate (all from GIBCO); recombinant mouse IL-2 (200 units/ml), human TGF-β1 (0.25 ng/ml, or as indicated in figure legends of individual figures), and anti-CD28 (37.51) (1.5 μg/mL) (Biolegend).
Th0 cultures were prepared by culturing 0.2 × 106 sorted T cells/well with 0.1 × 106 antigen presenting cells (APCs) in the presence of anti-CD3 (145–2C11) (1 μg/ml) (Biolegend) using Click’s media (Millipore-Sigma) containing 10% FBS (Gemini) and penicillin-streptomycin, L-glutamine, β-mercaptoethanol, HEPES, and sodium pyruvate (all from GIBCO) for 1–3 days. The APCs used in these cultures were isolated from pooled lymph node and spleen cells depleted of T cells (CD4+, CD8+, CD3+) and CD49b+ NK cells using biotinylated antibodies (Biolegend) and magnetic microbead selection (Miltenyi).
Th0 cultures were prepared by culturing 0.2 × 106 sorted T cells/well with 0.1 × 106 antigen presenting cells (APCs) in the presence of anti-CD3 (145–2C11) (1 μg/ml) (Biolegend) using Click’s media (Millipore-Sigma) containing 10% FBS (Gemini) and penicillin-streptomycin, L-glutamine, β-mercaptoethanol, HEPES, and sodium pyruvate (all from GIBCO) for 1–3 days. The APCs used in these cultures were isolated from pooled lymph node and spleen cells depleted of T cells (CD4+, CD8+, CD3+) and CD49b+ NK cells using biotinylated antibodies (Biolegend) and magnetic microbead selection (Miltenyi).
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