Easysep cd4 t cell negative selection kit

For isolation of CD4⁺ T cells, single-cell suspensions of splenocytes from 4T1 tumor-bearing control mice (day 16 after inoculation) were isolated by an Easysep CD4⁺ T cell negative selection kit (StemCell Technologies, Vancouver, BC, Canada). For isolation of MDSCs, single-cell suspensions of splenocytes from indicated groups were firstly incubated with Gr1-PE (listed in Supplementary Table 1) and then isolated with a PE positive selection kit (StemCell Technologies, Vancouver, BC, Canada).

For isolation of CD4⁺ and CD8⁺ T cells, spleens from the treated mice and 4T1 tumor-bearing control mice were harvested on day 21 after treatment, and single cell suspensions were prepared using a GentleMACS dissociator (Miltenyi Biotec). CD4⁺ and CD8⁺ T cells were isolated by an Easysep CD4⁺ T cell negative selection kit and an Easysep CD8⁺ T cell negative selection kit (StemCell Technologies, Vancouver, BC, Canada), respectively. CD4⁺ and CD8⁺ T cells with a purity of >90% were used for experiments. For isolation of Tregs, the isolated CD4⁺ T cells were then incubated with CD25-PE (clone 3C7, Biolegend, USA) and isolated with a PE positive selection kit (StemCell Technologies, Canada). The isolated cells were then verified by flow cytometry, and more than 85% of cells expressed Foxp3.