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Sample Preparation for Microinjection

there, it dries quickly and creates a blockage. Therefore, the filled capillary should be inserted into the capillary holder and immediately lowered into the medium to avoid blockage due to drying. The capillary should always remain in the medium especially during long experiments when large numbers of cells are injected. In order that the open petri dish has a sufficient pH stability under the microscope, a carbonate-free medium must be used (e.g., HEPES, pH 7.4 buffered, 10 mmol/l). A 3 cm dish should be filled with at least 2 ml medium. Cultivation can still take place in the usual medium containing carbonate. The speed and efficiency of automated systems allows biochemical analysis of microinjected cells. Between 500 and 1000 labeled cells are sufficient to analyze labeled proteins by gel electrophoresis. Cultivation and Microinjection
Preparation [3]
  1. Plate 250 cells in 5 l droplets in the center of a glass coverslip (10 x 10 mm).
  2. Place coverslips into a humid chamber and incubate at 37C until cells attach to the glass (usually takes 68 hr).
  3. Transfer coverslips into 35 mm petri dishes containing 2 ml of culture medium and let cells grow for 2 days at 37C. After this time, 500 to 1000 cells will usually be in the center of the coverslip.
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