This protocol provides a simple and reliable method for preparing a single-cell suspension from 5 - 10 mouse lungs. The resulting single-cell suspension is compatible with downstream applications, including EasySep™ cell isolation, which enables simple, column-free enrichment of target cell populations. Depending on the target cell type and downstream application (e.g. magnetic cell isolation, flow cytometry, cell culture, etc.), modifications such as red blood cell lysis or buffer selection may be required. For recommended sample preparation guidelines, refer to the EasySep™ kit-specific Product Information Sheet (PIS).

Materials

• Collagenase/Hyaluronidase (Catalog #07912)
• DNase I Solution (1 mg/mL, Catalog #07900)
• RPMI 1640 Medium (Catalog #36750)
• Dulbecco’s Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS with 2% FBS, e.g. Catalog #07905)
• 100 mm culture dish (e.g. Catalog #38045)
• Razor blade or scalpel
• 50 mL conical tube (e.g. Falcon® Conical Tubes, Catalog #38010)
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• 70 μm strainer (e.g. Catalog #27260)
• Syringe plunger
• Ammonium Chloride Solution (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 5 - 10 mouse lungs. If starting with more than 10 lungs, adjust volumes accordingly.

Part I: Mechanical Dissociation of a Mouse Lung Sample

1. Prepare 10 mL of dissociation 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 the medium. Mince into a homogenous paste (< 1 mm in size) using a razor blade or scalpel.
4. Transfer the minced lung tissue and the dissociation medium to a sterile 50 mL conical tube. Rinse the dish with the remaining 5 mL of dissociation medium and add it to the tube with the minced tissue. Incubate at 37°C for 20 mins on a shaking platform.

Part II: Preparation of a Single-Cell Suspension

1. Place a 70 μm nylon mesh strainer over a 100 mm dish. Pre-wet the strainer with 5 mL of PBS containing 2% FBS. Transfer the digested lung tissue mixture into the strainer. Push the tissue through the strainer with the rubber end of a syringe plunger to obtain a cell suspension.
2. Place a new 70 μm nylon mesh strainer over a 50 mL conical tube. Pre-wet the strainer with 5 mL of PBS containing 2% FBS. Transfer the mixture into the strainer and filter the cell suspension. Rinse the strainer with recommended medium*.
3. Centrifuge at 300 x g for 10 minutes at room temperature with the brake on low. Carefully remove and discard the supernatant.
4. Add 20 mL of ammonium chloride solution to the cell pellet and mix gently. Incubate for 5 - 15 minutes at room temperature or on ice (see kit-specific PIS for details).
5. 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.
6. Gently tap the tube to disassociate the cell pellet. Resuspend cells in the recommended medium* at the required cell concentration for your downstream procedure. Check the EasySep™ kit-specific Product Information Sheet (PIS) for the recommended information depending on your downstream application.

*Recommended medium may vary depending on the 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.

If you have any questions, contact us at techsupport@stemcell.com.