2.5. Isolation of CD3+ T cells and naïve
CD4+ T cells
After 8-week-old C57BL/6J mice were anesthetized with isoflurane, the
spleen was taken out, and the spleen single-cell suspension was prepared
following the previous method (Flaherty and Reynolds, 2015), and the
spleen cells were treated following the standard protocol of Naïve
CD4+ T Cell Isolation Kit (19765A, STEMCELL) to
isolate naïve CD4+ T cells. Then they were cultured in
a plate coated with CD3 antibody (100339, Biolegend), and continued to
be cultured in the presence of CD28 antibody (102115, Biolegend) until
be used. For GFP mouse spleen CD3+ T cells, after
obtaining spleen single cell suspension in the same manner as above,
Biotin anti-mouse CD3 Antibody (100303, Biolegend) and Anti-Biotin
MicroBeads (5190902231, Miltenyi Biotec) were added successively
according to the product protocals. The LS column magnetic bead sorting
method was used to obtain CD3+ T cells with a purity
of more than 90%.