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