We are attempting to patch clamp (whole cell) freshly isolated porcine coronary and rat uterine SMCs. Shortly after pipette contact with the cell, whether a seal is formed or not, the cell changes shape and a "puddle" forms around it. We cannot maintain a 1gigaohm seal for more than a few seconds. I am new to patch-clamping within the past two months and would appreciate tips or guidance in the right direction!
The bath osmolarity is 298 and the pipette solution is 287 mOsm.
Could you include a picture of what the cell looks like after patching?
Dear Kalen,
Sounds like your cells are not in good health/shape following harvest/dissociation process. One possibility is that cellular membranes are damaged and the membrane potential is depolarised and therefore the cells are not able to cope with the stress and mechanical pressure generated by the gigaSeal formation and whole-cell patch clamp. I don't know the details of your procedure of harvesting of cells. My guess is that one way of improving the cell/membrane quality may be to give them extra time following isolation. Once you obtain better quality cells, if the problem persists than focus on your internal (pipette) solution.
Best wishes,
Refik
What kind of glass do you use for patch pipettes? Soda-lime, borosilicate, ...?
Albertino
Pipette tip and softness of touch must be considered. In my experience with bovine coronary artery cells, good cells are few from the beginning and to look for ideal one was not so easy. How much is your pipette resistance? I used high resistance pipettes of 10-20 Mohm. It will be difficult to patch with thick one with resistance less than 5 Mohm. Mechanical stress can kill. If you push the cell too much with the pipette, the pipette presses the cell to bottom cover glass when you suck or push and kilsl the cell.
According to my experience osmolarity of solutions should be same. Or pipette solution even should have a bit higher osmolarity. In case when osmolarity of bath solution is higher it is very difficult to make a seal and held cell.
Try changing pipette Osmolality to 310 - 320 and lower the bath osmolality to around 290.
Very naively : are your cells kept immersed in the bathing solution?
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