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

In This Article

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
1. 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.
Catheter
1. Make a catheter by inserting a 23 G needle into transparent plastic polyethylene 21 G tubing (inner diamete....

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Results

Flow cytometry diagram, cell sorting process, FSC-SSC analysis, immune cell identification.
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 intratrac.......