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.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation
2. Performing the Bronchoalveolar Lavage (BAL)
3. Collecting the Cellular and Noncellular Components of the BAL Fluid
4. Analysis of the Different Cell Types in the BAL Fluid by Flow Cytometry
NOTE: One possibility is to analyze the absolute and relative cellular composition of the BAL fluid by performing flow cytometry. The goal of this paper is to elaborate on the technique of BAL. Flow cytometry is a specialized technique on its own. It is recommended to read specialized papers on the flow cytometry technique. Antibodies coupled to a fluorophore that recognize surface antigens (see Table 1) specific to a particular cell type(s) are used. By using a gating strategy, it is possible to identify T cells, macrophages, dendritic cells, B cells, eosinophils, and neutrophils in the cell fraction of the BAL.
Table 1: Selection of Immune Cell Surface Antigens. This table provides a list of surface epitopes used to characterize the different cell types. Combinations of several markers will be required to reliably define a specific cell type.
Antigen | Cell type |
Cluster of differentiation 3 (CD3) | Expressed on T cells |
Cluster of differentiation 11c (CD11c) | High expression on most dendritic cells, but also on monocytes, macrophages, neutrophils, and some B cells. |
Cluster of differentiation 11b (CD11b) | Expressed on the surface of many leukocytes including monocytes, neutrophils, natural killer cells, granulocytes, and macrophages. |
SiglecF | Alveolar macrophages and eosinophils. |
MHCII | Normally found only on antigen-presenting cells such as dendritic cells, mononuclear phagocytes, and B-cells. |
CD19 | B-lymphocyte antigen |
Ly-6G | A marker for monocytes, granulocytes, and neutrophils |
Table 2. List of Controls to be Included. This table shows all necessary controls for the accurate interpretation of the obtained results.
Samples | ||||
Tube | Antigen-fluorophore to be added to cells | Antibody stock concentration (mg/mL) | Antibody dilution | Total volume (µL) |
Fixable viability dye | 0.2 | 1/1000 | 50 | |
CD11c | 0.2 | 1/800 | 50 | |
SiglecF | 0.2 | 1/100 | 50 | |
sample X | MHCII | 0.2 | 1/200 | 50 |
CD3 | 0.2 | 1/200 | 50 | |
CD19 | 0.2 | 1/200 | 50 | |
CD11b | 0.2 | 1/200 | 50 | |
Ly6G | 0.2 | 1/200 | 50 | |
Voltage controls | ||||
Tube | Antigen-fluorophore to be added to cells | Antibody stock concentration (mg/mL) | Antibody dilution | Total volume (µL) |
Unstained cells | / | / | / | 50 |
Single-stained cells | Fixable viability dye | 0.2 | 1/1000 | 50 |
Single-stained cells | CD11c | 0.2 | 1/800 | 50 |
Single-stained cells | SiglecF | 0.2 | 1/100 | 50 |
Single-stained cells | MHCII | 0.2 | 1/200 | 50 |
Single-stained cells | CD3 | 0.2 | 1/200 | 50 |
Single-stained cells | CD19 | 0.2 | 1/200 | 50 |
Single-stained cells | CD11b | 0.2 | 1/200 | 50 |
Single-stained cells | Ly6G | 0.2 | 1/200 | 50 |
Compensation controls | ||||
Tube | Antigen-fluorophore to be added to beads | Antibody stock concentration (mg/mL) | Antibody dilution | Total volume (µL) |
Unstained beads | / | / | / | 200 |
Single-stained beads | CD11c | 0.2 | 1/2000 | 200 |
Single-stained beads | SiglecF | 0.2 | 1/2000 | 200 |
Single-stained beads | MHCII | 0.2 | 1/200 | 200 |
Single-stained beads | CD3 | 0.2 | 1/2000 | 200 |
Single-stained beads | CD19 | 0.2 | 1/2000 | 200 |
Single-stained beads | CD11b | 0.2 | 1/400 | 200 |
Single-stained beads | Ly6G | 0.2 | 1/200 | 200 |
Table 3: Overview of the Lasers and Filters of the Flow Cytometer used in This Study.
Laser type | Filter setup | |
505 LP | 525/50 | |
Blue (488 nm) | 550 LP | 575/26 |
100 mW | 670 LP | 685/35 |
750 LP | 780/60 | |
violet 405 nm | 450/50 | |
100 mW | ||
red 633 nm | 660/20 | |
70 mW | 750 LP | 780/60 |
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 the cell gate, single cells were identified using forward and side scatter. In this last population, cells that were alive were identified. CD11chigh cells and CD11clow cells were then identified. In the CD11chigh population, dendritic cells and macrophages were identified based on MHCII and SiglecF expression, respectively. In the CD11clow population, T cells and B cells were identified based on CD3ε and CD19 expression, respectively. In the remaining cell population, neutrophils and eosinophils were identified based on CD11b and Ly-6G expression, respectively.
The authors have nothing to disclose.
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 |