Cell Subculture protocol

Cell Subculture protocol.

Junchul

 

1.       Remove and discard the spent cell culture media from the culture dish.

2.       Wash cells using a SF media (approximately 2 mL per 10 cm₂ culture surface area).  Gently add wash solution to the side of the dish opposite the attached cell layer to avoid disturbing the cell layer, and rock the vessel back and forth several times.

3.       Remove and discard the wash solution from the culture dish

4.       Add the pre-warmed Trypsin-EDTA to the side of the flask; use enough reagent to cover the cell layer (approximately 0.5 mL per 10 cm₂).   Gently rock the container to get complete coverage of the cell layer.

5.       Incubate the culture vessel at room temperature for approximately 2 minutes.  Note that the actual incubation time varies with the cell line used.

6.       Observe the cells under the microscope for detachment.  If cells are less than 90% detached, increase the incubation time a few more minutes, checking for dissociation every 30 seconds.  You may also tap the vessel to expedite cell detachment.

7.       When ≥ 90% of the cells have detached, tilt the vessel for a minimal length of time to allow the cells to drain.  Add the equivalent of 2 volumes (twice the volume used for the dissociation reagent) of pre-warmed complete growth medium.  Disperse the medium by pipetting over the cell layer surface several times.

8.       Transfer the cells to a 15-mL conical tube and centrifuge then at 1500 rpm for 2 minutes.  Note that the centrifuge speed and time vary based on the cell type.

9.       Resuspend the cell pellet in a minimal volume of pre-warmed complete medium and remove a sample for counting.

10.    Determine the total number of cells and percent viability using a hemacytometer, cell counter and Trypan Blue exclusion.

11.    Dilute cell suspension to the seeding density recommended for the cell line, and pipet the appropriate volume into new cell culture vessels, and return the cells to the incubator.