Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry
Transcript
Begin with a multi-well plate containing a single-cell suspension of mouse retinal myeloid cells, including microglia, monocytes, and neutrophils.
Microglia — or retinal phagocytic immune cells, exhibit fluorescence due to engulfed fluorophore-labeled bioparticles of fungal origin, preinjected intravitreally.
Centrifuge and add Fc-receptor antibodies, blocking non-specific interaction sites.`
Introduce a cocktail of fluorophore-labeled antibodies targeting CD11b, Ly6C, and Ly6G surface proteins, interacting with myeloid cells expressing them.
Remove unbound antibodies. Treat with propidium iodide, which permeates through damaged membranes, exclusively staining dead cells.
In flow cytometry, excite propidium iodide to exclude dead cells and identify the non-fluorescent live cells.
Illuminate the cells with a violet laser; excited live cells signify the CD11b-expressing myeloid cells.
Next, illuminate with a red laser, exciting the near-far-red fluorophore within CD11b-positive cells, indicating Ly6C-expressing monocytes and Ly6G-expressing neutrophils.
Excite the cells with a blue laser; the emission of green fluorescence from the remaining CD11b-positive cells confirms the microglial uptake of fluorescent particles.
