Protocol: T Cell Isolation and Activation from Mouse Spleen and Lymph Nodes

 

Protocol: T Cell Isolation and Activation from Mouse Spleen and Lymph Nodes

1. Tissue Harvesting and Cell Suspension Preparation

• Isolate all lymph nodes (LNs) and spleens from 7–8 mice.
• Place tissues in isolation buffer (500 mL PBS + 2.5 g BSA + 2 mL 0.5 M EDTA) in a 6 cm dish on ice.
• Complete step 2-6 within 1 hour to maintain cell viability.

2. Tissue Dissociation

• Grind spleens and LNs on a 70 µm strainer fitted into a 50 mL tube using the plunger of a 1 mL syringe.
• Place 4–5 spleens on one strainer, remaining spleens and all LNs on the second.
• Flush with 5 mL isolation buffer multiple times (≥4–5), alternating with grinding.
• Also flush isolation buffer from dish and rinse dish with 5 mL isolation buffer into the strainers.
• Final volume per tube ~30 mL.

3. Red Blood Cell Lysis

• Centrifuge tubes at 420 g for 8 min at 4–10°C.
• Discard supernatant and resuspend pellet in 2 mL ACK lysis buffer, incubate at RT for 5 min.
• Add 10 mL isolation buffer to neutralize, mix by gentle shaking.
• Centrifuge again at 420 g for 8 min at 4–10°C.

4. Cell Washing and Filtration

• Resuspend each pellet in 5 mL isolation buffer.
• Filter through a new 70 or 40 µm strainer into a new 50 mL tube.
• Wash original tubes with 5 mL buffer and filter through the same strainer.
• Combine contents of two old tubes into one new tube; final volume ~20 mL.

5. Cell Counting

• Mix 5 µL of cell suspension with 95 µL Trypan Blue.
• Count viable cells using a hemocytometer or automated counter.
• Simultaneously, centrifuge remaining cells for magnetic sorting.

6. T Cell Isolation with Miltenyi Pan T Cell Kit

• Resuspend pellet in 40 µL isolation buffer per 2×10⁷ cells.
• Add 10 µL Biotin-Antibody Cocktail per 2×10⁷ cells, mix and incubate on ice for 5 min.
• Add 30 µL buffer per 2×10⁷ cells, then 10 µL MicroBead Cocktail, incubate on ice 10 min.
• Pass through 70 or 40 µm strainer, wash old tubes with 3 mL buffer and filter as well.
• Place LS columns on magnetic stand, pre-wash with 3 mL buffer.
• Apply cell suspension (5×10⁸ cells per LC column), collect flow-through (T cells).
• Wash columns with 3 mL buffer, combine flow-through and count cells.
• Final yield: ~3–5×10⁸ T cells.
• Centrifuge and resuspend at 5–10×10⁶/mL in T cell culture medium with 1× IL-2.
  (RPMI-1640, 10% EV-free FBS, 1% Pen/Strep, 55 µM 2-ME, 25 mM HEPES, 0.1 mM NEAA, 1 mM Sodium Pyruvate)

7. T Cell Activation with Beads

• Prepare beads at 4×10⁷/mL; for 3×10⁸ T cells use 1×10⁸ beads.
• Vortex beads for 30 s to fully suspend.
• Mix 1 volume of beads with 1 volume of fresh T cell medium.
• Vortex briefly, place on magnetic stand for 5 min, discard supernatant.
• Resuspend beads in T cell suspension.
• Plate T cell + bead mixture into T25 flask (this is Day 0).
• On Day 3: replace media with fresh IL-2-containing medium, collect medium (with EVs).
• On Day 6: 420g 8min, collect pellet and medium.
• Pellet (cells + beads), resuspend in 5 mL medium, place on magnet 5 min.
• Collect cell supernatant (avoid touching beads), repeat wash with 1 mL medium if needed.
• Combine all collected supernatants, count and centrifuge (420 g, 8 min), wash once with PBS.
• Resuspend in PBS at 0.5×10⁸/mL and store at –80°C.

Materials and Reagents

- Miltenyi Pan T Cell Kit (130095130), T Cell Activation Beads (11453D), LS Columns (130042401), IL-2

By Xuehao Zhang

05/22/2025