Dissociation of Mouse Lung Tissue into a Single-Cell Suspension

This is an example protocol for preparing a single-cell suspension from mouse lungs using enzymatic tissue digestion. The resulting cell suspension may be used for a variety of downstream applications, such as immunomagnetic cell isolation of a particular cell type.

Depending on your specific downstream application, your optimal lung dissociation protocol may differ from the procedure described below.


Materials


Protocol

The following instructions are for processing 5 - 10 mouse lungs. If starting with more than 10 lungs, adjust volumes accordingly.

  1. Prepare 10 mL of digestion medium by adding 1 mL of Collagenase/Hyaluronidase and 1.5 mL of DNase I Solution (1 mg/mL) to 7.5 mL of RPMI 1640 Medium. Warm to room temperature (15 - 25°C).
  2. Harvest lung tissue into a tube containing PBS with 2% FBS.
  3. Transfer lung tissue into a dish without medium. Mince into a homogenous paste (< 1 mm in size) using a razor blade or scalpel.
  4. Transfer minced lung tissue into a tube containing 10 mL of digestion medium and incubate at 37°C for 20 minutes on a shaking platform.
  5. Place a 70 μm nylon mesh strainer over a 100 mm dish and push the digested lung tissue through the strainer with the rubber end of a syringe plunger to obtain a cell suspension.
  6. Place a new 70 μm nylon mesh strainer over a 50 mL conical tube and filter the cell suspension. Rinse the strainer with recommended medium*.
  7. Centrifuge at 300 x g for 6 minutes at room temperature with the brake on low. Carefully remove and discard the supernatant.
  8. Add 20 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 6 minutes at room temperature with the brake on low. Carefully remove and discard the supernatant.
  10. Resuspend cells in recommended medium* at the required cell concentration for your downstream procedure.

*Recommended medium may vary depending on downstream application. As an example, for cell separation using EasySep™ Mouse ILC2 Enrichment Kit, the recommended medium is EasySep™ Buffer (Catalog #20144) or PBS (e.g. Catalog #37350) with 2% FBS and 1 mM EDTA. Medium should be free of Ca++, Mg++, and biotin.

  • Document #PR00044
  • Version 1.0.0
  • May 2021


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