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

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

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

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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.

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Fluorescent Dyes

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

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Technical Challenges

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