Entering the bath
a. Strongly
recommend to suck off some bath solution (to get rid of dust and contamminant)
b. Apply a small positive pressure.(10cmH2O)when the pippette enters the bath.Once the electrode is in the bath, don t bring out it again.
c. Then the Rmembrane will
be 2-10 Mohm. If Rm is too higher, there may have problems listed below:
(1) a bubble in the pipette,
(2) faulty connection to the probe input,
(3) bath electrode not connected,
(4) dusty things in the pippette.
If Rm is much lower than expected,say 100Kohm, the pippette may have been
broken.
a. After the pipette is in the
bath, you can release the pressure entirely before forming a seal. Somebody
likes to keep the tube in his mouth when he moves the pipette to the cell
surface. You can either keep the pressure on as you touch the cell surface,
or you can release the positive pressure first.
b. Once the pippette touches the cell surface, the situation can be very fluid. Perhaps it ll seal immediately, perhaps nothing will happen so you must push the pippette harder against the cell. Some cells like muscle or oocytes can take a lot of abuse, but neurons are delicious so you can t push so hard and be careful.
c. Once the pipette is on the cell, release the pressure first and give the cell a chance to seal itself. Wait for 30 second,then try gentle and continuous suction. As a rule, never apply suctions more than neccessary, or you will get short-lived seals or break into the cell. (You can try several brief pulses of suction rather than prolonged suction.) If a seal almost form, switch your amplifier to voltage clamp and apply a negative potential (say, -40 mV) to the pippette, it will help in establishing the seal.
d. The angle of the approach should not be a critical factor as you try to make the record pippette as perpendicular as possible to the cell surface.Usually an angle of at least 45 degree is adequate.You may find that the angle of approach affects how much pressure you must apply to form a seal.
e. When the pippette reaches the cell, typically the resistance will increase 1.5 (or 2 ) fold. Then apply a gental suction will result in the formation of gigaseal . To verify, set the GAIN to 50mV/pA, even then you'll only see the flat trace except for the capacitive spikes.
g. For manual manipulators,
try to make movement as smooth as possible. The actual movement of contact
between pippette and cell should be a single smooth movement without need
for further adjustment after the initial placement. As you place the electrode
on the cell, you have to judge how much pressure to apply to the cell at
the same time. Otherwise, if you make adjustments after the initial contact
you will disturb the process of seal formation and reduce your chances of
obtaining a GigaOhm seal.
3.Preparation
a. The time between media change
is believed to affect patching access. Recommend to change the media the
night before you experiment. So you can get fresh cells.
b. Vibration is you enemy. So make sure you experiment table is pressurized correctly. Too much or little air pressure will prevent vibration damping. All equipment should be attached to the surface of the air table. All loosened wires and tubes should be taped down. In addition, put your rig away from the air flow and foot traffic.
c. Sterile filter all you solutions
using 0.22 micron filter. Dusts, bacteria and other contaminants will surely
prevent seal formation. Also, check your solution pH value occasionally
because bacteria can sometimes start to grow in them.
d. Somebody has experience in having the best result with heavily polished
pipettes. It seems to be useful.
Forming a gigaseal
Preparation
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