Optimized Immunomagnetic Bead-based Negative Selection Protocol for CD4 T-cell Isolation from Mouse Lymph Nodes and Spleen

This study describes an immunomagnetic bead-based methodology for optimal purification of mouse CD4⁺ T cells. It reproducibly yields highly pure CD4⁺ T cells from mouse lymph nodes (95-99%) and spleen (93-96%) with no residual antigen-presenting cell (APC) function in the purified population. The recovery of the starting CD4⁺ T-cell population is consistently high (>70%) and many samples can be simultaneously processed in a short period of time. The key factors responsible for improved purity are combinations of monoclonal antibodies that were found, through trial and error, to yield T cells of maximal purity achievable by non-flow sort-based negative selection. These cocktails efficiently target unwanted cell subsets with antibodies against multiple surface markers expressed by non-CD4⁺ T cells. Because immunomagnetic bead-based protocols do not require the expensive and cumbersome processes required by flow sort-based purification, the methodology described here should find widespread use.