Cell Isolation HLA T Cell

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Upstream tips
Donor lymphocytes isolated as T and B cells (EasySep HLA T Cell Enrichment Kit and EasySep HLA B Cell Enrichment Kit, StemCell Technologies, Vancouver, Canada) were added separately to the wells of a Terasaki plate (1 × 105 1 μL buffer) together with 1 μL recipient sera.
Protocol tips
The plate was incubated for 40 minutes at 20°C to 25°C followed by a further 70 minutes after adding 5 μL rabbit complement (Bio-Rad).
Downstream tips
Then the samples were visualized with 3 μL of eosin and fixed with 5 μL of formalin. The result was acquired by visual inspection under a phase-contrast microscope.
Upstream tips
Mouse primary T cells were isolated from lymph nodes, and human T cells were isolated from the blood of healthy volunteers following university policies (MACSprep HLA T cell isolation kit, human).
Downstream tips
They were cultured in RPMI medium (containing 2 mM glutamine, 1 mM pyruvate, nonessential amino acids, and 50 μM 2-mercaptoethanol) supplemented with 10% FBS and 1% penicillin-streptomycin.
Protocol tips
Human CD3+ T cells were isolated from fresh whole donor blood in K2EDTA tubes using RosetteSep HLA T Cell Enrichment Cocktail according to instructions of the manufacturer (Stemcell).
Downstream tips
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