Mouse Lung Preparation for Flow Cytometry: Generating Cell Suspension from Lung Tissue for Flow Cytometric Analysis

Protokół

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

1. 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.
2. Soak the carcass in 70% ethanol and secure the mouse on a dissection board using tape.
3. 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.
4. 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.
5. 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).
6. Incubate the lung pieces 30 - 60 min at 37 °C and with constant shaking.
7. 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.
8. 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.
9. 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.

Materiały

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