T cell isolation

T cell isolation and purification from secondary lymphoid organs                            Jacobelli Lab

1. Retrieve donor mouse and euthanize according to institutional policies;
2. Separately harvest lymph nodes (we typically take inguinal, axillary, brachial, maxillofacial, and mesenteric) and spleen;
3. Place lymph nodes in a sterile mesh filter (40-100um mesh) (e.g. Corning Sterile Cell Strainer) in R10 media (RPMI + 10% FCS + Pen/Strep/Glut + BME) and smash through the filter with the back of a syringe piston;
4. Place the spleen in a separate sterile mesh filter in ammonium chloride (175mM NH₄Cl) and smash through the filter with the back of a syringe piston; ***Note, if you will be using the StemCell EasySep T cell isolation kit in step #7 below you should omit the red blood cell lysis step (#5) since the kit contains a red blood cell depleting antibody. Simply use R10 media instead of ammonium chloride for this step.
5. Lyse red blood cells in the spleen suspension for 3-5 min and then centrifuge (5 min at 400g) and resuspend with R10 media;
6. Count lymph node and spleen cells (you should expect ~100x10⁶ nucleated cells from the spleen and at least 30x10⁶ cells from pooled lymph nodes);
7. To purify CD8 or CD4 T cells use the appropriate negative selection kit from StemCell (e.g. EasySep Mouse CD4+ T Cell Isolation Kit, Catalog # 19852 ) following the manufacturer’s instructions.

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