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