Overview
This video describes the preparation of mouse lung cell suspension for flow cytometric analysis to study PD-L1 expression of lung adenocarcinoma cells.
Protocol
All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Lung Preparation for Flow Cytometry
- At the desired experimental endpoint, sedate the mouse by a subcutaneous injection of a mixture of ketamine (100 mg/kg of body weight) and xylazine (10 mg/kg of body weight) and euthanize it by cervical dislocation.
- Soak the carcass in 70% ethanol and secure the mouse on a dissection board using tape.
- Make a ventral midline incision and gently invert the skin to expose the thoracic wall muscles and the abdominal organs. Puncture the diaphragm and cut the ribs with scissors to expose the thoracic cavity.
- Perfuse the lungs 3x with 6 - 8 mL of ice-cold PBS through the right ventricle using a 27 G needle after cutting a small opening in the left ventricle to allow blood to leave. The lungs should be cleared of blood and turn completely white.
- Take out the lungs and mince the lobes into small pieces using scissors. Transfer the lung pieces to a 2 mL microcentrifuge tube and incubate it in 1.5 mL of lung digestion buffer (RPMI, 5% FCS, 150 U/mL collagenase I, and 50 U/mL DNase I).
- Incubate the lung pieces 30 - 60 min at 37 °C and with constant shaking.
- Transfer the lung cell suspension through a 70 µm cell strainer into a 50 mL tube. Clear the strainer with the back of a sterile 10-mL syringe and rinse the strainer with 15 mL of PBS with 2% FCS.
- Centrifuge the cells at 300 x g for 5 min at 4 °C and aspirate the supernatant. Resuspend the cells in 1 mL of ammonium-chloride-potassium (ACK) lysing buffer and incubate them for 5 min at room temperature for the lysis of residual erythrocytes.
- Centrifuge the cells at 300 x g for 5 min at 4 °C and resuspend the cells in 1 mL of PBS with 2% FCS and proceed with the desired staining protocol for flow cytometry
NOTE: Alternatively, the cells may be resuspended in RPMI containing 30% FCS and 10% DMSO and frozen using a freezing container for later analysis.
Subscription Required. Please recommend JoVE to your librarian.
Materials
| Name | Company | Catalog Number | Comments |
| Mouse lung adenocarcinoma cell line | isolated in-house | ||
| C57Bl/6 mice | F1 of the cross of the two backgrounds may be used (8-12 weeks) | ||
| 129S mice | |||
| RPMI 1640 Medium | Life Technologies | 11544446 | |
| Fetal Calf Serum | Life Technologies | 11573397 | |
| Penicillin/Streptomycin Solution | Life Technologies | 11548876 | |
| L-Glutamine | Life Technologies | 11539876 | |
| Ketasol (100 mg/mL Ketamine) | Ogris Pharma | 8-00173 | |
| Xylasol (20 mg/mL Xylazine) | Ogris Pharma | 8-00178 | |
| Blunt forceps | Roboz | RS8260 | |
| Student Iris Scissors | Fine Science Tools | 91460-11 | |
| DNase I (RNase-Free) | New England Biolabs | M0303S | |
| Collagenase Type I | Life Technologies | 17100017 | |
| ACK Lysing Buffer | Lonza | 10-548E |
