An 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.
Kamala
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Section on T Cell Tolerance and Memory (Ghost Lab), LCMI, NIAID, NIH, Rockville Pike, Bethesda, MD 20892, USA. tkamala@niaid.nih.gov
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology*; D018189:Immunomagnetic Separation / Q000379:methods*; D008198:Lymph Nodes / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D013154:Spleen / Q000166:cytology*
D000911:Antibodies, Monoclonal