ILC2s were sorted by magnetic beads combined with flow cytometry from mice spleen. Mice splenic cell suspension was stained by Biotinbinding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, antiCD3, anti-CD11b, and anti-CD49) (Miltenyi Biotec, Belgish-Gladbach, Germany), and then combined with Streptavidin-MicroBeads, the negative cells were enriched by magnetic column. This step was mainly to remove most of the lineage-expressing cells in spleen, thus enriching lineage-negative cells. The enriched cells were stained with FITC-lineage (eBioscience, San Diego, CA, USA), PE-CD90.2, and APC-ST2 (R&D Systems, Inc., Minneapolis, MN, USA), then further sorted by flow cytometry. The sorted cells (1 × 105 cells per well in a 96-well plate) were cultured in 1640 medium supplemented with 10% FBS, 10 ng/mL IL-7, 10 ng/mL IL-2, and10 ng/mL IL-33 (Peprotech, Rocky Hill, USA), 10 U/mL Penicillin and 10 U/mL streptomycin. Three days later, ILC2s were cultured with IgG-NPs at 0, 2, 4, 8 and 16μg/mL for 24h and 48 h. The cell viability was determined using cell counting kit (CCK8) (Dojindo, Japan) according to the manufacturer’s instructions. The plates were scanned at 450 nm for absorbance using a spectrophotometer (BioTek, Winooski, VT, USA). Each data point was measured for the average from six duplicates. The experiments were repeated independently for 3 times.
Anti cd45r
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD45R is a reagent used for the detection and analysis of CD45R expression on cells. CD45R is a protein tyrosine phosphatase that is expressed on the surface of various types of leukocytes. This reagent can be used in flow cytometry applications to identify and characterize cell populations expressing CD45R.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 7 (il 7), by Thermo Fisher Scientific (4 mentions)
Anti cd11b, by Miltenyi Biotec (4 mentions)
Anti ter 119, by Miltenyi Biotec (4 mentions)
Il 33, by Thermo Fisher Scientific (3 mentions)
Anti cd21 cd35, by Miltenyi Biotec (3 mentions)
Anti cd49, by Miltenyi Biotec (3 mentions)
Anti cd3, by Miltenyi Biotec (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Anti cd3 and anti cd28, by BioLegend (2 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Fitc lineage, by Thermo Fisher Scientific (1 mentions)
Anti mouse nk1, by BioLegend (1 mentions)
Facsmelody, by BD (1 mentions)
Anti tcrαβ, by Miltenyi Biotec (1 mentions)
Rb6 8c5, by BioLegend (1 mentions)
Anti cd11c, by Miltenyi Biotec (1 mentions)
Anti gr1, by Miltenyi Biotec (1 mentions)
M1 70, by BioLegend (1 mentions)
4 protocols using anti cd45r
1
ILC2 Isolation and Viability Assay
2
Isolation and Culture of Mouse ILC1s
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ILC1s were isolated from the spleens of C57BL/6 mice by combining magnetic beads and flow sorting. Mouse spleen cells were used to generated single-cell splenocyte suspensions[24 ]. The obtained cells were incubated with biotin-conjugated antibodies (anti-CD3ε, anti-CD45R, anti-Gr-1, anti-CD11c, anti-CD11b, anti-Ter119, anti-TCR-αβ, and anti-FCεRI; Miltenyi, Belgish, Germany) to enrich lineage-negative cells following the Miltenyi magnetic bead cell isolation protocol. Enriched lineage-negative single cell suspensions were stained with an anti-mouse lineage cocktail (BioLegend, 145-2C11, RB6-8C5, RA3-6B2, Ter-119, and M1/70), anti-mouse CD127 (BioLegend, A7R34), anti-mouse NK1.1 (BioLegend, PK136), and anti-mouse NKp46 (BioLegend, 29A1.4) for flow cytometry sorting (BD FACS Melody) of lineage− CD127+ NK1.1+ NKp46+ cells (which were considered ILC1s); these cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 0.5 ng/ml IL-7 (Gibco, PMC0071).
3
Isolation and Culture of Murine CD8+ T Cells and ILC2s
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Murine CD8 + T cells were isolated from tumor tissue using the tumor in ltration CD8 + T-cell magnetic bead sorting kit (Miltenyi Biotec, Belgish-Gladbach, Germany). Cell purity was con rmed by ow cytometry (96%). Sorted CD8 + T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco, NY, USA), 2 µg/ml anti-CD3 and anti-CD28 (Biolegend), 10 U/ml penicillin and 10 U/ml streptomycin (Gibco).
ILC2s were sorted from murine spleen by a combination of microbeads and ow cytometry. Brie y, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by ow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
ILC2s were sorted from murine spleen by a combination of microbeads and ow cytometry. Brie y, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by ow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
4
Isolation and Expansion of Murine CD8+ T Cells and ILC2s
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Murine CD8 + T cells were isolated from tumor tissue using the tumor infiltration CD8 + T-cell magnetic bead sorting kit (Miltenyi Biotec, Belgish-Gladbach, Germany). Cell purity was confirmed by flow cytometry (96%). Sorted CD8 + T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco, NY, USA), 2 µg/ml anti-CD3 and anti-CD28 (Biolegend), 10 U/ml penicillin and 10 U/ml streptomycin (Gibco).
ILC2s were sorted from murine spleen by a combination of microbeads and flow cytometry. Briefly, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by flow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
ILC2s were sorted from murine spleen by a combination of microbeads and flow cytometry. Briefly, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by flow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
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