Identification and Enumeration of Immune Cells in Bronchoalveolar Lavage Fluid by Flow Cytometry

Abstract

Source: Van Hoecke, L. et al., Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration. J. Vis. Exp. (2017)

This video demonstrates the use of flow cytometry to identify and count various immune cells in bronchoalveolar lavage fluid. Initially, single immune cells are identified using a viability dye, followed by differential staining of surface marker proteins, enabling the precise enumeration of each cell type.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation Lavage fluid Prepare a balanced salt solution with 100 µM ethylenediaminetetraacetic acid (EDTA). NOTE: To measure protein levels in the bronchoalveolar Lavage (BAL) fluid, it is recommended to add protease inhibitors to prevent protease activity in the BAL fluid.</l…

Representative Results

Figure 1: Gating Strategy for the Flow Cytometric Detection of Macrophages, Dendritic Cells, T Cells, B Cells, Neutrophils, and Eosinophils in BAL Fluid. BAL cells were isolated using the described BAL protocol. Cells were isolated from mice 24 h after intratracheal instillation of lipopolysaccharide. Counting beads and cells were identified based on forward- and side-scatter properties. In …

Materials

Balanced salt solution	Thermo Fisher Scientific	14175-129
Ethyleendiaminetetra acetic acid	Sigma-Aldrich	E6511	Irritating
23G x 1 1/4 needle	Henke Sass Wolf	4710006030	size: 0,60 x 30 mm
26G x 1/2 neelde	Henke Sass Wolf	4710004512	size: 0,45*12 mm
Plastic tubing	BD medical technology	427411	Polyethylene Tubing, I.D 0.58 mm (.023") O.D. .965 mm (0.38") 30.5m (100')
Sodium pentobarbital	Kela NV	514
Phosphate buffered saline	Lonza	BE17-516F	PBS without Ca++ Mg++ or phenol red; sterile filtered
1ml syringes	Henke Sass Wolf	5010.200V0	non pyrogenic and non toxic
Forceps	Fine Science Tools GmbH	91197-00
Surgical scissors	Fine Science Tools GmbH	91460-11
Centrifuge tube 50 ml	TH.Geyer	7696705	Free from Rnase/Dnase/endotoxin
Centrifuge tube 15 ml	TH.Geyer	7696702	Free from Rnase/Dnase/endotoxin
Microcentrifuge tube 1,5 ml	Sigma-Aldrich	0030 120.094	Polypropylene
Microcentrifuge	Sigma-Aldrich	5415R
Centrifuge	Thermo Fisher Scientific	75004030
Ammonium-chloride-potassium (ACK) lysing buffer	Lonza	10-548E	Sterile filtered
Live/dead -efluor506	ebioscience	65-0866-18	Fixable viability dye
CD11c-PE-cy7	eBiosciences	25-0114-81
SiglecF-PE	BD Pharmingen	552126
MHCII-APCefluor780	Biolegend	107628
CD3-PE-cy5	VWR	55-0031-U100
CD19-PE-cy5	eBiosciences	15-0193-83
CD11b-V450	BD Pharmingen	560455
Ly6G-AF700	Biolegend	127621
Absolute Counting Beads	Life Technologies Europe B.V.	C36950
anti-CD16/CD32	BD Pharmingen	553142
96-well 340 µl storage plate plate	Falcon	353263	V-bottom, natural polypropylene
Flow cytometer	BD Biosciences
Catheter	BD Biosciences	393202