Lum's Notes

Seeding density recommended for the cell line：
6CM培养皿0.5-1million、5ml 1 million 5ml;10cm-2million-10ml ;25ml-2million-10ml DMEM培养基

All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.

1. Remove and discard the spent cell culture media from the culture vessel.
2. Wash cells using a balanced salt solution without calcium and magnesium (approximately 2 mL per 10 cm2 culture surface area). Gently add wash solution to the side of the vessel opposite the attached cell layer to avoid disturbing the cell layer, and rock the vessel back and forth several times.
   注： The wash step removes any traces of serum, calcium, and magnesium that would inhibit the action of the dissociation reagent.
3. Remove and discard the wash solution from the culture vessel
4. Add the pre-warmed dissociation reagent such as trypsin or TrypLE™ 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 200 × g for 5 to 10 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 growth 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, or the Countess Automated Cell Counter. If necessary, add growth media to the cells to achieve the desired cell concentration and recount the cells.
    注： We recommend using the Countess Automated Cell Counter to determine the total number of cells and percent viability. Using the same amount of sample that you currently use with the hemacytometer, the Countess Automated Cell Counter takes less than a minute per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells. See the protocol on Counting Cells with a Hemacytometer.
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.
    备注： If using culture flasks, loosen the caps before placing them in the incubator to allow proper gas exchange unless you are using vented flasks with gas-permeable caps.