BioSample

Protocols: HeLa/Fucci(SA)2 cells (RCB2867, Riken BRC) were maintained in Dulbecco’s Modified Eagle Medium (Nacalai Tesque) containing 10% fetal bovine serum (gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). We employed a micromanipulation system integrated with an inverted microscope, featuring two micromanipulators: PatchMan NP2 (Eppendorf) and TransferMan 4r (Eppendorf) to position a glass capillary (HUMANIX, CT-0P) proximity to a cellular membrane. One micromanipulator was equipped with a capillary holder (Pipette holder, Narishige, H-7), positioned at a 75° angle. The glass capillary was filled with 1 μL of sucrose solution (260 mM Sucrose and 20 mM HEPES) at the tip as an inner solution. A platinum wire of 200-μm in diameter (Niraco, PT-351265) was immersed in the sucrose solution. The another manipulator was equipped with a platinum electrode of 1 mm in diameter (Niraco, PT-351481). The platinum electrode immersed in a sucrose mixture medium approximately 5 mm away from the microscope's field of view. Each platinum wire was linked to a source meter (Keithley, 2410-C) for real-time monitoring of current values and application of the voltage via an RS-232 C serial communication. To detect the contacting of a tip of the glass capillary to the cellular membrane, a constant voltage of 0.3 V was maintained before the sampling. The contact was detected by damping of the ionic current. Upon contact, intracellular products were collected into the capillary using square pulse conditions of 2 Hz, 2 V spacing, -10 V pulse, and 10% duty ratio for 5 s for HeLa/Fucci cells. Subsequently, the capillary was swiftly withdrawn from the medium and detached from the capillary holder to collect the extracted RNA.