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Functional Analysis of Ionic Flux in Isolated Endosomes

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.

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Functional Analysis of Ionic Flux in Isolated Endosomes

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

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

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

  4. 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.

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

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

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