hTERT HME2

46,XX (6 cells)
45,XX, der(10;15)(q10;q10)

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 to 400 x g for 8 to 12 minutes.
4. Carefully aspirate the supernatant and discard, leaving the cell pellet.
5. Gently resuspend the cell pellet with the appropriate amounto of complete growth medium and transfer cell suspension into a vented T-75.
6. Place the flask in a 37°C incubator with 5% CO₂ .

It is recommended to subculture at less than 90% confluence. Do not allow cultures to become over confluent.

Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with PBS to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA for Primary Cells (ATCC PCS-999-003) to flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 33°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of Trypsin Neutralizing Solution (ATCC PCS-999-004) and aspirate cells by gently pipetting.
5. Centrifuge the cell suspension at approximately 150 to 400 x g for 8 to 12 minutes to remove dissociation agent.
6. Resuspend the cell pellet in an appropriate amount of complete culture medium.
7. Add appropriate aliquots of the cell suspension to new culture vessels.
   Cultures can be established between 2.0 x 10⁴ and 4.0 x 10⁴ viable cells/cm².
8. Incubate cultures at 37°C.

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