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Eaton Lab Innovation & Conventional patch clamping

Eaton Lab Innovation & Conventional patch clamping. cell. Methods: Voltage clamp (single channel patch clamp analysis). The patch clamp set up. micromanipulator. Inverted microscope. Pipet holder. Images taken from http://www.sciencedisplay.com/patchclamp.html.

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Eaton Lab Innovation & Conventional patch clamping

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  1. Eaton Lab Innovation & Conventional patch clamping cell

  2. Methods: Voltage clamp (single channel patch clamp analysis) The patch clamp set up micromanipulator Inverted microscope Pipet holder Images taken from http://www.sciencedisplay.com/patchclamp.html

  3. Pipet is approaching cell of interest

  4. MONITOR current Forth coming contact of the pipet tip with the cell surface can be monitored with the oscilloscope time Send out a test (+20mV) current pulse

  5. MONITOR current To get a seal, tight contact of the pipet with plasma membrane, a soft suction has to be applied time

  6. MONITOR current Suction of the patch pipet causes deformation of the cell membrane and the building of a seal. time

  7. Na+ 2 2 1 1 N C C MONITOR current Ion channels in the cell membrane can be investigated. time • All proteins are in their natural environment. • The patch is so small a single channel can be investigated.

  8. Na+ N- terminus N- terminus 2 2 1 1 N C C N The Eaton Lab investigates: Ion channels: Primary cells: Cell lines: Human lung ENaC (sodium) Xenopus kidney, murine kidney, chinese hampster ovary, murine alveolar lung…. Rat lung -Highly selective -Non-selective Murine lung CFTR (chloride)

  9. Eaton Lab Innovation 1. Novel In situ patch clamp 1.Anesthetize and sacrifice animals in accordance with Institutional Animal Care and Use Committee. 2. Perfuse lungs to remove intravascular blood elements. 3. Agarose fill lungs to stabilize delicate tissue. 4. Section 250mm slices; horizontal plane across the middle lobes.

  10. Helms et al -VP (mV) GNa =4.4pS -80mV I (pA) -60mV -40mV -VP (mV) +20mV GNa =13.5pS 0mV I (pA) 1pA -20mV 5s Figure2. A. B. Highly Na+ Selective Channel (HSC) C. Nonselective Cation Channel (NSC)

  11. Eaton Lab Innovation 2. Novel Identification of CFTR chloride channels in lung cells. pipet Single channel recording with 100mM amiloride back-filled in pipet

  12. Eaton Lab Innovation +O2 SODi CTR 15mM NONOate 2mM NONOate 3. Complex drug delivery protocols. Continual patch clamp recording in one A6 cell with multiple drug treatments. control + superoxide dismustase inhibitor + low concentration nitric oxide + high concentration nitric oxide increase O2-

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