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

Published: January 31, 2024

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 …

Offenlegungen

The authors have nothing to disclose.

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

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Identification and Enumeration of Immune Cells in Bronchoalveolar Lavage Fluid by Flow Cytometry. J. Vis. Exp. (Pending Publication), e21914, doi: (2024).

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