Functional Analysis of Ionic Flux in Isolated Endosomes

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Functional Analysis of Ionic Flux in Isolated Endosomes Michael Carrithers Department of Neurology

Can voltage-gated ionic flux enhance acidification of macrophage endosomes during infection?

Goal: Use flow cytometry on isolated endosomes to complement microscopy and electrophysiology.

Experimental Approach Differentiate and prime a monocytic cell line (THP-1). Isolate endosomes on a sucrose gradient. Dialyze sample. Load with functional fluorescent dyes. Activate channel. Analyze on MoFlo. Collect enriched endosomal fraction.

Fluorescent Dyes Lysosensor Green (pH) Sodium Green CoroNa Red Disbac2(3) (slow voltage-sensitive dye)

Technical Challenges Particle size (200-800 nm) Pressure Sheath buffer Large amount of starting material