When approaching the pipette to form a seal, very precise micromanipulators are required. RBCs are “designed” for passing through small capillaries. When passing through the spleen, RBCs have to go through tiny slits whose VX809 mean size has been recently measured at 1.89 μm in length and 0.65 μm in width.43 Therefore, patch-pipette tips must be rather thin, with an opening smaller than 1 μm (corresponds to roughly 10–15 MΩ in physiological saline solutions) to avoid the entry of the cell into the pipette. Besides the pipette size, its shape has to be adapted such that a piece of membrane enters the

pipette for seal formation without totally entering into the pipette when depression (typically 20 mbar) is applied. The pipette tip must be thin enough, but at the same time tapered enough, to preserve a low electrical access resistance. Another

issue arises from RBC’s high deformability. GSK1120212 The portion of the RBC membrane that enters into the pipettes during seal formation varies. Furthermore, it has been recognised that membrane deformation induces transient Ca2 + entry in RBCs.73 Such transient activity may generate secondary transient anionic channel activity.74 This phenomenon leads to a change in the intracellular K+ concentration that has to be taken into account for data interpretation. Therefore, the time of seal formation and calibrated depression must be mentioned in publications. The small RBC size results in a small membrane capacitance of

approximately 1–1.3 pF.[75] and [76] This becomes relevant during the transition from the cell-attached to whole-cell configuration. The rupture of the membrane fragment inside the pipette tip is typically achieved by a brief electrical pulse (200 ms, 500 mV). A successful whole-cell configuration can be checked via the sudden appearance of membrane capacitance transient currents, which can be easily compensated on the amplifier. Acetophenone Nevertheless, the situation is different in plate-based “pipettes” as they are used by automated patch-robots (Fig. 3). There, the basal capacitance of the plate is much higher and an increase of 1 pF is almost invisible. Therefore, the major indication for reaching the whole-cell state is the increase in current, which is a challenge because differentiation between the loss of seal resistance and the whole cell current needs to be probed in the experimental protocol. However, if the seal resistance is approximately 10 GΩ, the current leakage at + 100 mV can be calculated to be 10 pA, presenting a relation to Ohm‘s law. Typical whole-cell recordings show current values between 200 and 1000 pA or even higher, which often are rectifying, i.e., they do not follow Ohm‘s law; then, the leak remains below 1–5% of the total current.