The protocol below is an example for generating a single-cell suspension from solid tumors from mouse models of breast cancer (4T1), melanoma (B16-F10), and colon cancer (CT26.WT), but it may be applicable to a variety of other tissues. The resulting cell suspension can be used for a variety of downstream applications, such as the isolation of tumor-infiltrating leukocytes (TILs) using EasySep™ Mouse TIL (CD45) Positive Selection Kit.

Materials

• Collagenase/Hyaluronidase (Catalog #07912)
• DNase I Solution (1 mg/mL, Catalog #07900)
• RPMI 1640 Medium (Catalog #36750)
• 35 mm culture dish (e.g. Catalog #38069)
• Razor blade, scalpel, or scissors
• 14 mL round-bottom tube (e.g. Falcon® Round-Bottom Tubes, Catalog #38008)
• 70 μm strainer (e.g. Catalog #27260)
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• 50 mL conical tube (e.g. Falcon® Conical Tubes, Catalog #38010)
• Ammonium chloride solution (e.g. Catalog #07800)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
• Serological pipette controller
• Serological pipettes (e.g. Falcon® Serological Pipettes, 10 mL, Catalog #38004)
• Recommended medium (may vary depending on downstream application)*
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Protocol

The following instructions are for processing ≤ 1 g of tumor tissue. For > 1 g of tumor tissue, adjust volumes accordingly.

1. Prepare 5 mL of tumor digestion medium by combining the following:
   • 500 μL Collagenase/Hyaluronidase
   • 750 μL DNase I solution (1 mg/mL)
   • 3.75 mL RPMI 1640 medium
   Mix thoroughly and warm to room temperature (15 - 25°C).
2. Harvest the tumor tissue into a dish.
3. Mince the tumor tissue into small pieces (≤ 2 mm) using a razor blade, scalpel, or scissors.
4. Transfer the minced tumor tissue to a 14 mL round-bottom tube containing tumor digestion medium (prepared in step 1).
5. Incubate at 37°C for 25 minutes on a shaking platform.
6. Place a 70 μm nylon mesh strainer on a 50 mL conical tube and rinse with recommended medium*. Transfer the digested tumor tissue into the strainer. Using the rubber end of a syringe plunger, push digested tumor tissue through the strainer. Rinse the strainer with recommended medium, then top up the tube to 50 mL with recommended medium*.
7. Centrifuge at 300 x g for 10 minutes at room temperature with the brake on low. Carefully remove and discard the supernatant.
8. Add 10 mL of ammonium chloride solution to the cell pellet. Incubate at room temperature for 5 minutes.
9. Top up to 50 mL with recommended medium*. Centrifuge at 300 x g for 10 minutes at room temperature with the brake on low. Carefully remove and discard the supernatant. Resuspend cells at 5 x 10^7 cells/mL in recommended medium.

Note: For higher TIL purity, resuspend cells at 5 - 10 x 10^7 cells/mL; for higher TIL recovery, resuspend cells at 1 - 2.5 x 10^7 cells/mL in recommended medium.

*Recommended medium may vary depending on downstream application. For cell separation using EasySep™ Mouse TIL (CD45) Positive Selection Kit, the recommended medium is EasySep™ Buffer (Catalog #20144) or PBS (e.g. Catalog #37350) containing 2% FBS and 1 mM EDTA. Medium should be free of Ca++ and Mg++.