In vivo cell depletion

1.   Validated reagents for in vivo cell depletion

• Anti-asialo GM1 (αAGM1, 014-09801, FujiFilm Wako Pure Chemical Corporation), 20 μg/dose
• Rabbit IgG isotype control (Thermo Fisher Scientific), 20 μg/dose
• Anti-CD200R3 (Ba103; Hycult Biotech, Uden, the Netherlands), 30 μg /dose
• Rat IgG2b isotype control (RTK4530; BioLegend), 30 μg /dose
• Anti-Ly6G (1A8; BioLegend), 200 μg/dose
• Rat IgG2a isotype control (RTK2758; BioLegend), 200 μg/dose
• Clodronate liposome (Hygieia Bioscience), 50 mg/kg
• Control liposome (Hygieia Bioscience), 50 mg/kg
   

2.   Reagents for flow cytometery (All from BioLegend, 1/200 dilution except for secondary antibody)

• NK cells
  APC-Cy7 anti-CD3 (145–2C11) and PerCP-Cy5.5 anti-NK1.1 (PK136)
• Basophils
  PE anti-c-Kit (2B8) and APC anti-CD49b (DX5)
• Macrophages
  PE anti-F4/80 (BM8) and APC anti-CD11b (M1/70)
• Neutrophils
  Ly-6G (1A8), FITC Goat anti-rat IgG (1/500 dilution), and PE anti-CD11b (M1/70)

3.   In vivo depletion protocol

• Dilute reagents in sterile PBS and keep on ice until use.
• Inject 200 µL of antibody solution intraperitoneally on the first and/or second day before tumor cell injection.
• For αAGM1, two doses before tumor inoculation give better depletion efficiency.
• For some experiments that need cell depletion for two weeks, inject antibody on Day -1, 0, and 7 post tumor injection.

4.   Validation of the depletion efficiency

• Dissect lungs and/or spleen from the mice. 
  a. Preparation of a single-cell suspension of the lung cells
  i. Mince the resected lungs with scissors and incubate the tissue in 10 mL of RPMI containing 200 U/mL collagenase type IV (Lakewood) and 5 U/mL DNase I (Roche) for 30 min at 37°C.
  ii. Place a ϕ40-μm cell strainer (BD Bioscience) on a 50 mL tube.
  iii. Transfer the cell suspension to the strainer placed in the 50 mL tube.
  iv. Centrifugation at 500×g for 5 min at 4℃.
  v. Resuspend with 10 mL of PBS.
  vi. Centrifugation at 500×g for 5 min at 4℃.
  
  b. Preparation of a single-cell suspension of the splenocytes
  i. Place a ϕ40-μm cell strainer on a 10 cm dish containing 10 mL of PBS.
  ii. Put a spleen on the cell strainer.
  iii. Gently mash the spleen with the rubber end of a plunger of a 5 mL syringe.
  iv. Place a ϕ40-μm cell strainer on a 50 mL tube.
  v. Transfer the cell suspension to the 50 mL tube.
  vi. Centrifugation at 500×g for 5 min at 4℃.
  vii. Remove the strainer, discard the supernatant, and add 1 mL of ACK lysing buffer (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA).
  viii. Leave the cell suspension for 2 min on ice.
  ix. Add 10 mL PBS.
  x. Transfer the cell suspension in a ϕ40-μm cell strainer placed on a 50 mL tube.
  xi. Centrifugation at 500×g for 5 min at 4℃. 
   
• Cell surface staining (NK cells, basophils, and macrophages)
  i. 3×10⁵ cells were suspended with 50 μL of PBS containing 2 % BSA and diluted antibodies.
  ii. Leave the cell suspension for 20 min in 4 ℃.
  iii. Add 1 mL of PBS containing 2 % BSA.
  iv. Centrifuge at 500×g for 5 min at 4℃.
  v. Resuspend with 300 uL of PBS containing 2 % BSA.
  vi. Analyze by flow cytometry.
   
• Intracellular staining (Neutrophils)
  • Resuspend 3×10⁵ cells with 100 μL of BD cytofix/cytoparm solution (BD Bioscience) and leave for 15 min at 4 ℃.
  • Add 1 mL of BD perm/wash buffer (BD Bioscience).
  • Centrifuge at 800×g for 5 min at 4℃.
  • Resuspend cells with 50 μL BD perm/wash containing Ly6G antibody.
  • Leave the cell suspension for 20 min at 4 ℃.
  • Add 1 mL of BD perm/wash buffer and centrifuge at 800×g for 5 min at 4℃.
  • Repeat the wash three times.
  • Resuspend with 50 μL BD perm/wash buffer containing FITC Goat anti-rat IgG antibody for 20 min at 4℃.
  • Add 1 mL BD perm/wash buffer and centrifuge at 800×g for 5 min at 4℃.
  • Repeat the wash three times.
  • Resuspend with 50 μL BD perm/wash buffer with anti-CD11b antibody for 20 min at 4℃.
  • Add 1 mL of BD perm/wash buffer solution and centrifuge at 800×g for 5 min at 4℃.
  • Repeat the wash three times.
  • Resuspend with 300 μL of PBS containing 2 % BSA.
  • Analyze by flow cytometry.

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