Microelectrode holder half cell

The 900A micropressure system (World Precision Instruments) was used to make direct measurements of intracellular pressure according to the manufacturer’s instructions and as described in Petrie and Koo (2014) (link). A 0.5-µm micropipette (World Precision Instruments) was filled with a 1-M KCl solution, placed in a microelectrode holder half-cell (World Precision Instruments), and connected to a pressure source regulated by the 900A system. A calibration chamber (World Precision Instruments) was filled with 0.1 M KCl and connected to the 900A system, and the resistance of each microelectrode was set to zero and then secured in a MPC-325 micromanipulator (Sutter Instrument) within an environmental chamber (37°C and 10% CO₂) on an Axiovert 200M microscope (ZEISS). To measure intracellular pressure, the microelectrode was driven at a 45° angle into the cytoplasm, maintained in place for ≥5 s, and removed. The pressure measurement was calculated as the mean pressure reading during this interval of time. In individual polarized, elongated cells, a pressure measurement was first made behind the nucleus, followed by a measurement in front of the nucleus in the same cell.

Direct measurements of intracellular pressure were performed using the 900A micropressure system (World Precision Instruments) according to the manufacturer’s instructions as described previously (Petrie and Koo, 2014 (link)). Briefly, a glass micropipette with a 0.5-µm-diameter tip (World Precision Instruments) was filled with a 1-M KCL solution, placed in a microelectrode holder half-cell (World Precision Instruments), and connected to a pressure source regulated by the 900A system. The calibration chamber (World Precision Instruments) was filled with 0.1 M KCl and connected to the 900A system, and the resistance of each microelectrode was set to 0 and attached to a MPC-200 micromanipulator (Sutter Instrument) within an environmental chamber (37°C and 10% CO₂) on an LSM 700 microscope (Zeiss). To measure intracellular pressure, the microelectrode was driven at a 45° angle into the cytoplasm, maintained in place for ≥5 s, and removed. The pressure measurement was calculated as the mean pressure reading during this time interval. The experimenters were blinded to the identity of the five cell lines to eliminate potential bias.