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

Abstract

Source: Moll, H. P., et al. Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression. J. Vis. Exp. (2019).

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

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 organ....

Materials

List of materials used in this article

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