Splenic CD3+ T cells were immunopurified from Satb1f/f mice (Mouse T Cell negative selection kit, 19851; StemCell Technologies) and labeled with the proliferation tracker Cell Trace Violet as indicated. T cell proliferation was stimulated by adding mouse specific anti-CD3/CD28 dynabeads (ThermoFisher) at a 1:1 T cell to bead ratio according to the manufacturer’s protocol. CD3+CD4+FoxP3GFP natural Treg cells (nTreg cells) were sorted from both CD4CreSatb1f/fFoxP3GFP and Satb1f/fFoxP3GFP control littermates. Immunopurified CD3+ T cells (2×105) were subsequently co-cultured at 1:2, 1:4, 1:8, and 1:16 T cell to nTreg cells ratios and incubated for 3 days prior to flow cytometry analysis.
Mouse t cell negative selection kit
Manufactured by STEMCELL
Sourced in Canada
The Mouse T Cell Negative Selection Kit is a laboratory tool designed to isolate mouse T cells from a mixed cell population. The kit utilizes a magnetic-based separation technique to selectively remove non-T cells, leaving the desired T cell population. This allows for the enrichment and purification of T cells for downstream applications.
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2 protocols using mouse t cell negative selection kit
1
Functional Assessment of Treg Cells
2
Purification and Characterization of T Cell Subsets
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Pooled spleens and lymph nodes were collected from WT or Plac8-/- mice and processed by gently pressing them through a 70 μm filter. Where noted, CD4 and CD8 T cells were subsequently purified using a mouse T cell negative selection kit (Stemcell Technologies, Vancouver, Canada) according to manufacturer’s instructions. T cells were stained for 15 min at 4°C in PBS + 0.1% BSA (Gemini Bio-Products) using anti-mouse antibodies purchased from eBioscience and Tonbo Biosciences (both San Diego, CA): CD16/CD32 (93), CD4 (IM7), CD8 (53–6.7), CD44 (IM7), CD62L (MGL-14), CD45RB (C363.16A), and CD25 (PC61.5). Propidium iodide (Sigma-Aldrich) was used to exclude dead cells (PI+) from the sorted population. For T cell in vitro assays, live cells were designated as CD4+ or CD8+ T cells before sorting out naïve T cells (CD62L+, CD44lo) or memory T cells (CD44hi). For the T cell transfer model of colitis, live CD4+ T cells were sorted as (CD25-, CD45RBhi) effector cells.
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