This article details a method for analyzing immune cell populations in mouse bronchoalveolar lavage fluid using flow cytometry. The protocol includes staining cells with fluorescent antibodies to identify specific immune cell markers.
Begin with a multi-well plate containing mouse bronchoalveolar lavage fluid comprising an immune cell population stained with a green fluorescent viability dye.
Introduce Fc-antibodies to block immune cells' Fc-receptors.
Add a cocktail of distinct fluorophore-labeled antibodies targeting specific cell markers. Remove unbound antibodies and resuspend the cells in a buffer.
Using flow cytometry, identify dim fluorescence singlets, indicating live cells.
Illuminate cells at 488 nanometers, causing cells to emit red fluorescence correlating with CD11c-expressing cells.
Within the high CD11c population, with appropriate wavelength filters, identify cell marker proteins MHC-II on dendritic cells and SiglecF on macrophages.
In the CD11c low population, expression of CD3 on T cells and CD19 on B cells enable their identification.
Illuminate cells at 405 nanometers, emitting blue fluorescence, signifying the CD11b-expressing neutrophils.
Illuminating cells at 633 nanometers excites far-red fluorophores on Ly-6G-expressing eosinophils.
Using flow cytometry data, enumerate the abundance of each type of immune cell in the bronchoalveolar lavage fluid.
After the third bronchoalveolar lavage collection, centrifuge the pooled aspirate, and store the supernatant at negative 80 degrees Celsius. Resuspend the pellet in 200 microliters of ACK lysis buffer. After no more than two minutes at room temperature, dilute the lysis buffer with one milliliter of cold PBS and collect the cells by centrifugation.
Resuspend the pellet in the appropriate volume of PBS for the number of samples to be analyzed and aliquot the cells into the appropriate number of wells of a 96-well plate for the analysis. Collect the cells by another centrifugation, and resuspend the pellets in 50 microliters of FC block in PBS.
After 10 minutes at room temperature, add 50 microliters of the appropriate antibody cocktail of interest to each well, and incubate the cells for 30 minutes at four degrees Celsius protected from light. At the end of the incubation, centrifuge the plate and discard the supernatant, resuspending the pellets to a final volume of 200 microliters of FACS buffer per well for flow cytometric analysis.
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