Hmm my last post didn't post right, so i'll have to abbreviate out of frustration of typing twice :)

-Check the drift yourself, the way you mount your pipette to the holder can influence whether or not it drifts. If you apply too much tension to a screw/o-ring configuration for example there will be drift as the o-ring relaxes.

-Check your angle of approach. If you are patching from anywhere but perfectly vertically above the cell you should not be patching into the middle - you want the surface of your pipette tip to be tangent to the membrane. This might explain the cell's movement as the top-end of your pipette 'pulls' the cell outward

-Check how far you are going into the cell. If you are pushing too deeply into the cell, I've seen patches rupture on their own as you describe. After getting a seal, back your pipette off so that is *just* touching the surface of the cell.

Thank you very much Jonny.

I am a rookie so any suggestion or caution is always welcome!

I will check the drifting again. It's just frustrating that two months ago, everything is smooth and perfectly fine. And all of the sudden everything up side down.

My patching angle is around 45 degree. Usually which part of the cell should I patch?

SMC are usually pretty flat but I don't think I pushing it too deeply usually as long as I see about 10% increase in resistance I stop and apply suction and I did not have any problem in getting giga-seal. but just cannot get to whole cell configuration or stable whole cell configuration. 

I'm not sure it is my growing condition or I should not plate the cell 12 h before I patch?

Yes! Patching is the art of managing frustration, hold out for the 1/1000 time where the stars align and the holy grail of good data peeks through the clouds.

If you are patching around 45 degrees, you'll want to patch wherever on the cell is also 45 degrees! If the cell is round-ish, that'll be about 1/3 of the way in (looking from above). If the cells are flatter, you'll want to go more towards the edge.

Do you have the blessing of visual guidance? (you mention guiding by resistance?)

Hmm, my approach style is typically 1)use a large-diameter pipette (1.5-2x diameter of patch pipette) to clean the surface of the cell with light positive pressure, 2)approach with positive pressure in patch pipette, decreasing as I reach the surface of the cell. 3)When I see dimpling, I slowly creep forward while slowly releasing positive pressure until I think my pipette is where the surface of the cell would rebound to 4)release pressure but *don't apply suction yet.* allowing the membrane to settle on the tip of the pipette by itself typically gives the best seals, once you see the resistance start to climb up around 60-80MOhm then *very light* suction should seal the deal. Perhaps your immediately applying suction causes membrane trauma - might explain the self-rupturing patches?

I typically patch in slice, so I'm not much help with culture techniques, sorry :\

One more thing - you might want to let the cell get used to its new fate of a pipette stuck to its forehead before trying to break through. I let the cell set 3-4m after getting the GOhm seal, then break through with light, percussive suction.

You mention that your primary "SMC are usually pretty flat".  We use acutely dissociated vascular SMCs and the cells appear like small nematode worms with axial ratios of between 5:1 and 10:1.  Such cells are quite easy to seal on.  As primary cells "sit" in culture (in your case for at least 12 hours), they spread out, begin de-differentialting into a proliferative phenotype, and the surface membrane becomes more stretched and fragile.  

Thank you very much Dave. Now I can break in to the cell. But cannot maintained a stable seal >10 min. Because I need to do dose response, it makes me impossible to do it.  I also found that either the cell roll over/ the pipette shifted but the pipette was moved from within the cell boundary to the edge of the cell and then lost the seal.

I would love to get isolated cell but unfortunately, we don't have access to those. I have to rely on primary cell culture from ATCC. I

s there any trick?

Kin, from what you mention re "cell roll over/the pipette shifted...", it seems that you should go back to checking the problem of either pipette or stage drift.  Get a stage micrometer and position a patch pipette over a mark just about a micro above the micrometer surface.  Note the pipette position in three dimensions.  Let setup stand for 30 minutes and then come back and compare with the position with 30 min before. Especially check the vertical distance. Any change will indicate relative movement in your setup that could account for your loss of seal.

How is your perfusion set up? If the flow is too high it could cause instability as well - this is especially important since you are doing a dose-response, rate of flow can affect things like binding constants.