KOSEN 한인과학기술자네트워크

닫기

더 많은 혜택을 위해
로그인 하세요.

지식나눔

지식나눔

patch clamp 문의

2014-03-05 org.kosen.entty.User@37fa1f0d 황병문(arim14)
  • 1
patch clamp 실험을 하는 중 seal test (0) 화면에서 구리선(?)을 만져도 저항값의 변화가 없습니다.
배지에 구리선을 담가도 저항값의 변화가 없음.
주위에 물어보니 직사각형으로 생긴 기기( 초코바 정도의 크기, 실험실에 있는데 이름을 모르겠음)를 이용하여 고치는 것이라고 함. 이 기기를 구리선 대신 기계에 연결하여 교정한다고 함.
하지만 나는 기기는 연결할 줄 알지만, 컴퓨터의 clamp program 에서 어떻게 조작해야 하는지 모르겠음. ( seal test, membrane test???)--주위에 정확히 아는 사람이 없습니다.
현재 patch clamp 8.0 Demo 를 사용중.
혹시 패취클램프 연구 중 컴퓨터의 clamp program의 seal test (0) 화면에서 전선을 만지거나 배지에 담가도 seal resistance 에 변화가 없는 경우,
이를 해결하는 방법을 아시는 분은 답을 달아주시면 감사하겠습니다.
전선이 끊어지거나 다른 이유는 아닌것으로 생각됨.

 
  • patch clamp
지식의 출발은 질문, 모든 지식의 완성은 답변! 
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
답변하기
답변 1
  • 답변

    이배훈님의 답변

    2014-03-06
    • 0

    첨부파일

    참고바랍니다. (online trouble shooting)
     
    ROUBLESHOOTING
    I cannot obtain GΩ seals
    1. Make sure your preparation is healthy and has always been oxygenated; check the pH and osmolarity of your ACSF and filling solution.
    2. Make sure you are placing the electrode in an area of high cell density.
    3. Check the shape of your microelectrode tip. Keep the resistance in the right range (4–6 MΩ for mature neurons, 8–12 MΩ for small cells).
    4. Check the pressure line to the microelectrode holder for leaks.
    5. Clean the microcapillaries and make sure that you are not contaminating them with oils while handling.
    obtain a GΩ seal but cannot “break in” because I lose the seal when trying
    1. Check the pressure line to the microelectrode holder for leaks or obstructions.
    2. Try the “zap” function in your amplifier.
    3. Try a different microelectrode. Lowering the resistance may help.
    My seals do not last very long
    1. Once in whole-cell mode, apply light positive pressure.
    2. Check the shape of your microelectrode tip.
    3. Make sure your preparation is healthy.
    4. Check the osmolarity of the solutions.
    5. Make sure the ACSF bath and drug application system does not have air bubbles> Make sure no vibration of the stage or microelectrode holder.
    I am not sure whether I am patching the right cell type
    1. Make sure your preparation is healthy.
    2. Be familiar with the basic electrical properties of your cell of interest.
    3. Include a dye to track cell morphology post hoc.
    I am not sure whether my preparation is healthy
    1. Perform a rapid cell-death and survival assay using representative preparations and the fluorescent markers propidium iodide (dead cells) and Syto-11 (live cells).
    Does age of the animal matter?

    Yes. up to P12, it is durable. After P12, it becomes increasingly difficult.

    My electrode measures 7MΩ in the saline, but as soon as I exert pressure, it goes up to 15MΩ. Should I patch?

    No. Your solution contains dust which clogs the tip of the electrode. If this happens 3 times repeatedly, refilter your pipette solution.

    My electrode is >50MΩ with visible capacitance!! help!

    You have bubbles in the tip of your pipette. Take the pipette out and give it a few taps.

    Does penetration angle matter?

    Probably Yes. In general, 45 degree or shallower makes better giga seals.

    I always get dendrites, but my boss needs somatic recording.

    Make a bigger patch electrode (e.g. 4-6MΩ) and start deeper in the brain (say, layer 2/3).

    What is the ideal tip resistance?

    It depends, but just to patch and establish current clamp recording, 5MΩ is probably the choice. Smaller (i.e. higher resistance) is slightly easier to form a seal, but more difficult to break in, and the access resistance will be bigger. Bigger (i.e. lower resistance) will get you to soma and probably good voltage clamp, but seal formation will be more difficult. I’ve never managed to make a seal with electrodes smaller than 3MΩ.

    How do I chloride my silver electrode?

    Put it in bleach for 15 – 30 min.

    Electrolysis in saline (150 – 300 mM NaCl) with 5 – 10V.

    Voltage shows slow DC shift, especially when I turn on step command. What should I do?

    It is likely that your electrode (either reference or electrode) is not chrolided. (i.e. silver is exposed, instead of AgCl, or silver is somehow oxidized). Put the silver wires in bleach for 15 min or so.

    I cannot exert pressure / suction. Why?

    Check your Tee junction.

    How much biocytin should I put in my internal solution?

    Again, the answer is it depends. Usually, 0.5% to 2% (w/v) is more than enough. Mind the osmolality. It should be around 290-310.


     

    Patch Clamp Protocol
     
    1. Mary Johnson Ph. D.
      mary at labome dot com
      Synatom Research, Princeton, New Jersey, United Stat
    답변수정
    참고바랍니다. (online trouble shooting)
     
    ROUBLESHOOTING
    I cannot obtain GΩ seals
    1. Make sure your preparation is healthy and has always been oxygenated; check the pH and osmolarity of your ACSF and filling solution.
    2. Make sure you are placing the electrode in an area of high cell density.
    3. Check the shape of your microelectrode tip. Keep the resistance in the right range (4–6 MΩ for mature neurons, 8–12 MΩ for small cells).
    4. Check the pressure line to the microelectrode holder for leaks.
    5. Clean the microcapillaries and make sure that you are not contaminating them with oils while handling.
    obtain a GΩ seal but cannot “break in” because I lose the seal when trying
    1. Check the pressure line to the microelectrode holder for leaks or obstructions.
    2. Try the “zap” function in your amplifier.
    3. Try a different microelectrode. Lowering the resistance may help.
    My seals do not last very long
    1. Once in whole-cell mode, apply light positive pressure.
    2. Check the shape of your microelectrode tip.
    3. Make sure your preparation is healthy.
    4. Check the osmolarity of the solutions.
    5. Make sure the ACSF bath and drug application system does not have air bubbles> Make sure no vibration of the stage or microelectrode holder.
    I am not sure whether I am patching the right cell type
    1. Make sure your preparation is healthy.
    2. Be familiar with the basic electrical properties of your cell of interest.
    3. Include a dye to track cell morphology post hoc.
    I am not sure whether my preparation is healthy
    1. Perform a rapid cell-death and survival assay using representative preparations and the fluorescent markers propidium iodide (dead cells) and Syto-11 (live cells).
    Does age of the animal matter?

    Yes. up to P12, it is durable. After P12, it becomes increasingly difficult.

    My electrode measures 7MΩ in the saline, but as soon as I exert pressure, it goes up to 15MΩ. Should I patch?

    No. Your solution contains dust which clogs the tip of the electrode. If this happens 3 times repeatedly, refilter your pipette solution.

    My electrode is >50MΩ with visible capacitance!! help!

    You have bubbles in the tip of your pipette. Take the pipette out and give it a few taps.

    Does penetration angle matter?

    Probably Yes. In general, 45 degree or shallower makes better giga seals.

    I always get dendrites, but my boss needs somatic recording.

    Make a bigger patch electrode (e.g. 4-6MΩ) and start deeper in the brain (say, layer 2/3).

    What is the ideal tip resistance?

    It depends, but just to patch and establish current clamp recording, 5MΩ is probably the choice. Smaller (i.e. higher resistance) is slightly easier to form a seal, but more difficult to break in, and the access resistance will be bigger. Bigger (i.e. lower resistance) will get you to soma and probably good voltage clamp, but seal formation will be more difficult. I’ve never managed to make a seal with electrodes smaller than 3MΩ.

    How do I chloride my silver electrode?

    Put it in bleach for 15 – 30 min.

    Electrolysis in saline (150 – 300 mM NaCl) with 5 – 10V.

    Voltage shows slow DC shift, especially when I turn on step command. What should I do?

    It is likely that your electrode (either reference or electrode) is not chrolided. (i.e. silver is exposed, instead of AgCl, or silver is somehow oxidized). Put the silver wires in bleach for 15 min or so.

    I cannot exert pressure / suction. Why?

    Check your Tee junction.

    How much biocytin should I put in my internal solution?

    Again, the answer is it depends. Usually, 0.5% to 2% (w/v) is more than enough. Mind the osmolality. It should be around 290-310.


     

    Patch Clamp Protocol
     
    1. Mary Johnson Ph. D.
      mary at labome dot com
      Synatom Research, Princeton, New Jersey, United Stat
    등록된 댓글이 없습니다.
목록
목록