Bronchoalveolar Lavage Fluid Collection from a Mouse Model Infected with Non-typeable Haemophilus influenzae

Samenvatting

Source: Venuprasad, K., et al. Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation. J. Vis. Exp. (2016)

This video demonstrates the collection and processing of bronchoalveolar lavage fluid from a euthanized mouse infected with non-typeable Haemophilus influenzae for downstream analysis.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Bronchoalveolar Lavage Fluid (BALF) Collection
   1. Sacrifice the mouse by cervical dislocation 24 hr after infection.
   2. Open the mouse's abdominal cavity with anatomical scissors, cut the ventral aorta to bleed, and expose the ventral part of the lung.
   3. Expose the trachea and intubate it with a 20-gauge catheter. Instill 0.8 ml of BALF buffer (1x PBS (Phosphate buffered saline), 0.1% dextrose, 10 U/ml heparin) and collect the lavage by gentle aspiration. Repeat this procedure three times.
   4. Count the total number of cells in 100 µl of BALF suspension using a hemocytometer under 10X magnification with Trypan Blue staining.
   5. Prepare slides for modified Giemsa staining. Aliquot 100 µl of BALF into the appropriate wells of the cytospin and centrifuge at 500 x g for 5 min. Dry the slides for 10 min and stain the cells using modified Giemsa stain according to the manufacturer's protocols.
   6. The remaining cells can be used for subsequent analysis, such as fluorescence-activated cell sorting (FACS), confocal microscopy, gene expression, and western blot.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Dextrose Anhydrous	Themo Scientific	R01300
Heparin	Hospira,Inc	RL-3010
Syringe needle 20/26G	BD	(REF305115/175)
1 ml syringe	BD	REF 309602
Catheter 20GA	BD	REF 381433
Tweezer #5 Stainless steel, 11cm long	kentscientific	INS600095
5ml Polystyrene round-bottom tubes	BD	REF 352058
1.5 ml Microcentrifuge tubes	Light Labs	A-7001-R
1X PBS (Phosphate buffered saline)	Gibco	10010-023
RBC Lysis Buffer (10X)	Biolegend	420301