Submitted by Lauren French (download pdf)

 

The following guidelines can be used to replate the cells:

 

T75 flask: Plate 0.75 - 1.8 x 106 cells/flask

 

T25 flask: Plate 0.25 - 0.625 x 106 cells/flask

 

35 mm dish: Plate 0.1 x 106 cells/dish

 

To Replate for Recording:

  • The afternoon prior to the day of recording, remove coverslips in tissue culture dishes from refrigeration (plastic or glass).
  • I remove all of the NGF-complete media from the T25 flask, and replace with 5 mls of NGF-complete media and gently blow-off the cells and lightly triturate to remove the long processes from the PC12 cells.
  • I place about 1 ml of cell suspension on each of 5 treated coverslips (that have been pre-scored).  Leave the dishes in the hood for about 15 min at RT to settle and attach onto the coverslips.
  • Then, add 1.5 ml of complete media to each of the 35 mm tissue culture dishes and return to the incubator.  (Note: I use a 150 mm plate to transport between the hood and incubator to reduce the likelihood of infection; once in the incubator, I move the 150 mm plate lid off to the side a bit to allow the air to equilibrate.)
  • Try different dilutions of cells depending on how densely the cells are growing in the flasks when replating to give the optimal density for recording.
  • Within a single day, there should be cells that can be located visually with the microscope that do not have processes more than 2 times the diameter of the cell body, and that remain completely isolated from any other cells.
  • Greater trituration of the cell suspension results in better separated clumps of cells into individual cells and cells with shorter processes.  I choose cells to record that are isolated and that are well-attached to the coverslip and have short processes leaving the cell body.
  • Avoid any fibroblast-appearing cells as the PC12 cells are not a pure culture.  Initially, I always try about 2-3 different dilutions of cells on coverslips treated with collagen.

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