CD3+ cells were isolated from at least 25 × 106 PBMCs. Peripheral blood mononuclear cells were thawed in defrosting medium (10% fetal calf serum in Dulbecco's phosphate-buffered saline), centrifuged at 1,300 rpm, RT, for 10 min, then counted and used for T-cell isolation by negative immunomagnetic depletion using the Pan T Cell Isolation Kit human (Miltenyi Biotec, Germany), consisting in a Pan Cell Biotin-Antibody Cocktail and a Pan T Cell MicroBead Cocktail that provide the retention of the unwanted cell fraction (CD3-negative cells) and the elution of the CD3+ cells fraction through the LS column placed on a suitable MACS Separator (Miltenyi Biotec). Mean CD3+ cells value per sample was 4 × 106 (±2.2).
Pan t cell microbead cocktail
Manufactured by Miltenyi Biotec
Sourced in Germany
The Pan T Cell MicroBead Cocktail is a laboratory tool designed for the isolation and enrichment of T cells from various sample types. It contains a mixture of magnetic beads coated with antibodies specific to surface markers expressed on T cells, allowing for their efficient separation and purification.
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2 protocols using pan t cell microbead cocktail
1
Isolation of CD3+ T cells from PBMCs
2
Adoptive Transfer of T Cells in PKCζ-KO Mice
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T cells were isolated from the spleens of WT or PKCζ-KO mice using the Pan T Cell Isolation Kit II according to the manufacturer’s instructions (Miltenyi Biotec). In brief, splenocytes were stained with Pan T Cell Biotin-Antibody Cocktail (Miltenyi Biotec), followed by addition of Pan T Cell Micro Bead Cocktail (Miltenyi Biotec) before separation using a MACS Separator (Miltenyi Biotec). After sorting, the purity of T cells was assessed by flow cytometry and confirmed to be ∼92–97% (data not shown). The 2 × 106 T cells isolated from WT of PKCζ-KO mice were transferred into each PKCζ-KO mouse intravenously. The number of transferred T cells (2 × 106 cells) was twice the number (106 cells) that had been previously shown to initiate T cell–dependent responses sufficiently in naive C57BL/6 mice (Shaw et al., 2015 (link)). 12 h after T cell transfer, mice were intravenously infected with 5 × 103P. berghei sporozoites.
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