Prepare reagents listed below.
- 1L cDMEM [1000 mL DMEM (1x), 100mL FBS (heat inactivated), 25 mL HEPES (1M), 10mL L-Glutamine (200 mM), 10 mL Pen/Strep. Filter the mix through 500 mL Bottle top Filter (0.2 μm aPES membrane)].
- 1L mTCM [1000 mL RPMI1640 (w/ 20 mM HEPES) (1x), 100 mL FBS (heat inactivated), 10 mL Sodium Pyruvate (100 mM), 1 mL HEPES (1 M), 10 mL Pen/Strep, 100 uL 2-Mercaptoethanol (0.5 M). Filter the mix through 500 mL Bottle top Filter (0.2um aPES membrane)].
- 500 mL EasySep Buffer [500 mL D-PBS (1x), 10 mL FBS (heat inactivated), 1 mL EDTA (0.5 M). Filter the mix through 500mL Bottle top Filter (0.2 μm aPES membrane)].
- 500 mL 2% BSA blocking solution [500 mL D-PBS (1x), 10 g dry weight Bovine Serum Albumin. Filter through 500 mL Bottle top Filter (0.2 μm aPES membrane)]. Combine and let dissolve for >15 minutes prior to filtering.
- 500 mL Flow Buffer [500 mL D-PBS (1x), 2.5 mL FBS (heat inactivated), 3.6 mL EDTA (0.5 M)]. No need to filter.
- Diluted Retronectin (12.5 μg/mL diluted in 1x D-PBS and aliquotes stored at -20C until use).
- 1000x human IL-2 stock (50,000 IU/mL in 1x D-PBS; reconstituted per manufacturer). hIL-2 is cross-reactive and is more economical.
- 1000x human IL-15 stock (10,000 IU/mL in 1x D-PBS; reconstituted per manufacturer). hIL-15 is cross-reactive and is more economical.