In vitro Treg cell suppression assays were performed as previously described (9 (link), 48 (link)). In brief, splenic CD4+CD25+ cells (~90% Foxp3+) were isolated using the EasySep Mouse CD4+CD25+ Regulatory T Cell Isolation Kit II (STEMCELL Technologies), incubated with a 1:1 ratio of MACSiBeads (Miltenyi Biotec) coated with anti-CD3/anti-CD28 and cocultured with differing ratios of Treg to CD4+ T effector (Teff) cells labeled with CellTrace Violet (Thermo Fisher Scientific), which were obtained from 8-week-old WT mice. After 48 hours, effector T cell proliferation was assayed by flow cytometry analysis.
Macsibeads
Manufactured by Miltenyi Biotec
MACSiBeads are magnetic beads used for the separation and enrichment of specific cell types and biomolecules in research and diagnostic applications. The beads are coated with antibodies or ligands that can bind to target cells or molecules, allowing their isolation or depletion from complex samples.
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Lab products found in correlation
Rhil 2, by Roche (3 mentions)
Golgistop, by BD (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Easysep mouse cd4 cd25 regulatory t cell isolation kit 2, by STEMCELL (1 mentions)
Golgiplug, by BD (1 mentions)
Rituximab, by Roche (1 mentions)
Facsverse, by BD (1 mentions)
Fixable viability stain 450, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Mouse th1 th2 th17 phenotyping kit, by BD (1 mentions)
Pma ionomycin, by BD (1 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Cd19 6d5, by BioLegend (1 mentions)
Cd24 m1 69, by BioLegend (1 mentions)
8 protocols using macsibeads
1
Treg Cell Suppression Assay Protocol
2
Purification and Activation of Mouse T Cells
3
Functional Assay of NK Cell Activation
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For functional assays, CD56+ MACS-enriched cells were thawed, FACS-purified for viable CD3− CD56+ NK cells, and rested overnight in complete medium. The next day, 2 × 10e5 NK cells were mixed with 1 × 10e5 target cells in V-bottom 96-well plates and the indicated concentrations of IL-12 and/or IL-18 were added. For cross-linking of CD16, rituximab (Roche) was present during the assay at the indicated concentrations. For sole engagement of NKG2C, MACSi beads (Miltenyi) were coated with an agonistic anti-NKG2C antibody (R&D Systems; biotinylated in-house) as previously described (11 (link)), and 1 × 10e6 αNKG2C beads were added to 2 × 10e5 NK cells. After 1 h incubation, GolgiPlug and GolgiStop (both BD Biosciences) were added according to the manufacturer’s instructions, and the assay continued for 5 h.
4
Magnetic Bead-Bound Activation Reagents
5
Activation and Phenotyping of Mouse T-cells
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Primary T-cells were isolated from 3-4 mouse spleens by negative selection on magnetic bids using the Pan T cell Isolation Kit II (Miltenyi Biotech), see Grum-Schwensen et al. for details [13 (link)]. Purified T-cells were maintained in RPMI for 3 or 6 days with anti-CD3 or a combination of anti-CD3 and anti-CD28 antibodies, coupled to MACSibeads (Miltenyi Biotec) plus 10 ng/ml recombinant IL2 as described in [28 (link)].
Activated T-cells were stimulated with S100A4 protein (1 μg/ml) or S100A4 protein mixed with 6B12 antibody (6 μg/ml). Before fixation, PMA/Ionomycin and Golgistop™ (BD Biosciences) were added for 5 hours. Cells were washed with PBS and Fixable Viability Stain 450 (BD Biosciences) was added to discriminate between viable and dead cells.
Cells were fixed using the Cytofix/Cytoperm™ kit (BD Biosciences) and stained with the mouse Th1/Th2/Th17 phenotyping kit (BD Biosciences) containing antibodies against CD4, IL17A, IFNγ and IL4 according to the manufacturer’s instructions. Data acquisition and analysis were performed on a FACSVerse (BD Biosciences) using FlowJo software (Tree Star). All experiments were repeated 3-5 times.
Activated T-cells were stimulated with S100A4 protein (1 μg/ml) or S100A4 protein mixed with 6B12 antibody (6 μg/ml). Before fixation, PMA/Ionomycin and Golgistop™ (BD Biosciences) were added for 5 hours. Cells were washed with PBS and Fixable Viability Stain 450 (BD Biosciences) was added to discriminate between viable and dead cells.
Cells were fixed using the Cytofix/Cytoperm™ kit (BD Biosciences) and stained with the mouse Th1/Th2/Th17 phenotyping kit (BD Biosciences) containing antibodies against CD4, IL17A, IFNγ and IL4 according to the manufacturer’s instructions. Data acquisition and analysis were performed on a FACSVerse (BD Biosciences) using FlowJo software (Tree Star). All experiments were repeated 3-5 times.
6
Isolation and Activation of Murine T Cells
7
Isolation and Activation of Murine T Cells
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T cells were purified from single-cell suspensions of spleen and lymph
nodes from male and female mice aged 6–12 weeks by negative selection
with biotinylated antibodies (CD8, CD19, B220, CD11b, CD11c, DX5, Ter119 and
CD24, UCSF Monoclonal Antibody Core) and magnetic anti-Biotin beads (MACSi
Beads, Miltenyi Biotec). For IL-2 stimulation, purified T cells were
pre-activated on 96-well plates coated with anti-CD3 (2C11) and anti-CD28
(37.51) for 72 hours, removed and cultured with rhIL-2 (100 u/mL, Roche) for 36
hours, and then cultured without rhIL-2 for the 36 hours prior to all
experiments.
nodes from male and female mice aged 6–12 weeks by negative selection
with biotinylated antibodies (CD8, CD19, B220, CD11b, CD11c, DX5, Ter119 and
CD24, UCSF Monoclonal Antibody Core) and magnetic anti-Biotin beads (MACSi
Beads, Miltenyi Biotec). For IL-2 stimulation, purified T cells were
pre-activated on 96-well plates coated with anti-CD3 (2C11) and anti-CD28
(37.51) for 72 hours, removed and cultured with rhIL-2 (100 u/mL, Roche) for 36
hours, and then cultured without rhIL-2 for the 36 hours prior to all
experiments.
8
T Cell Purification from Murine Tissues
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