Human Primary T Cells: A Practical Guide

Guide

• Live document: Human Primary T Cells: A Practical Guide
• Preprint (v1): https://peerj.com/preprints/26993/
• Protocols: https://www.protocols.io/view/human-primary-t-cells-a-practical-guide-quvdww6

Notebooks

• Hundreds of millions of T cells can be isolated from a buffy coat sample
• The relative abundance of T subsets vary across donors
• Activation is required to rapidly expand T cells in culture but cell counts barely increase throughout the activation process
• Electroporation is a feasible method for genetically manipulating T cells
  • Plasmid electroporation is inefficient and causes major cell death
  • mRNA electroporation is convenient for transiently expressing exogenous proteins
  • Cas9 RNP electroporation is a feasible technique to knockout a gene
• Common T cell techniques can be optimized for cost
  • Untouched T cell isolation kit contents can be titrated down without major efficiency loss
  • Using fewer anti-CD3 and -CD28 beads for activation doesn’t immediately compromise activated cell yield
  • IL2 supplement is not necessary for short-term T cell cultures during and after CD3/CD28-based activation
  • T cell isolation can be circumvented by culturing PBMCs with activation beads
  • NIH’s reagent service provides access to highly standardized and cost-effective recombinant IL2