• Centrifuge tubes
• Petri dishes, 35x10 mm (Fisher Scientific, Ottawa, ON. cat. no. 08-757-100A)
• Centrifuge capable of up to 600x g
• Inverted microscope
• 37 ºC Incubator with 5% CO2
• Surgical scissors
• Electric hair shaver
• Non-treated Tissue culture plates – 24 well (BD Bioscience, Mississauga, ON. cat. no. 351147)
• Suspension cell TC flasks T25/T75 vented (Sarstedt, Montreal, QC. cat. no. 83-1810-502 and 83-1813-502)
• Sterile round bottom falcon tubes, 12x75 mm (VWR, Mississauga, ON. cat. no. 734-0445)
• 70uM cell strainer (Fisher Scientific, Ottawa, ON. cat. no. 08-771-2)
• 10 mL Syringes (BD Bioscience, Mississauga, ON. cat. no. 309604)
• EasySep™ Magnet (StemCell Technologies, Vancouver, BC. cat. no. 18000)
• 1cc Insulin syringes (BD, Mississauga, ON. cat. no. 329420)
• Heat pad or heat lamp
• Anaesthesia and euthanasia apparatus
Dynabead wash buffer: Prepare ahead of time, Sterile PBS with 0.1% FBS and 2 mM EDTA, pH 7.4.
1x PBS and DPBS: Prepare ahead of time by diluting with sterile distilled H2O.
Complete medium AR10: Prepare ahead of time, Advanced RPMI 1640 with 10% FBS, 2mM GlutaMAX and 100 U/mL Penicillin-Streptomycin.
Mouse Recombinant IL-7: Reconstitute to 25 ng/µL in sterile PBS with 0.1% FBS. Aliquot and store at -20 ºC.
Mouse Recombinant IL-15: Reconstitute to 50 ng/µL in sterile PBS with 0.1% FBS. Aliquot and store at -20 ºC.
Complete medium AR10 supplemented with cytokines: Prepare fresh, AR10 supplemented with 30 U/mL human recombinant IL-2, 25 ng/mL mouse recombinant IL-7 and 50 ng/mL mouse recombinant IL-15.
Dynabead mixture: Prepare fresh, follow the manufacturer’s protocol to resuspend the dynabeads via vortex for 30 seconds. 25 µL of dynabeads is required for each 1 million LNCs. Pipette dynabeads into a sterile round bottom centrifuge tube and add 1 mL of dynabead wash buffer, pipette gently 30 times without generating air bubbles. Place the centrifuge tube into an EasySep magnet for 1 minute. Decant/Pipette the wash buffer into a waste container while keeping the tube inside the magnet; the dynabeads will adhere to the wall of the tube. Remove the tube from the magnet and add AR10 supplemented with cytokines at 500 µL/25 µL dynabeads. Make sure all of the dynabeads on the wall of the tube are reuspended into the medium.