2. AC duration: approx 60 to 99 s.
3. DC: approx 250 to 300 V (280 V). Adjust DC voltage according to gap distance between electrodes. The appropriate electric field strength is approx 2.5 to 3.0 kV/cm. When 2-mm gap Microslides are used, the DC should be around 600 V.
3.1.3. Setup of Microslide Chambers
1. Sterilize the Microslides by immersion in 70% ethanol followed by flaming.
2. Set a Microslide in a 100-mm plastic dish chamber made from a bacterial dish.
3. For each electrofusion, apply 40 |L of cell mixture into the 1-mm electrode gap on the Microslide.
4. Connect the Microslide with the Micrograbber cable to the ECM 2001 (Fig. 2B).
5. Place the chamber on an inverted microscope to check cell alignment and compression. It is important to determine the optimal fusion conditions under the microscope each time. The fusion conditions may vary depending on cell density and cell size.
Fig 2. (A) Setup of AC/DC pulse generator ECM2001 (BTX) (B) Microslide chamber in a 100-mm plastic dish made using a bacterial dish and Micrograbber cable. (C) Cell mixture of ES cells and thymocytes applied between electrodes. (D) Pearl chain formation during AC application. (E) Instruments for dissociating mouse thymus into single cells. (F) Culture dishes with inactivated PEFs prepared 1 d before cell fusion. (G) The recommended fusion parameters.
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