Contents 1 Materials 2 Protocol 3 Result 3.1 Observation

Thawing HeLa Cell Line Frozen Stocks

2014/8/4 Recover cells from frozen, start our cell cultrue experiment

[2014 Aug 4th]

Thawing HeLa cells, start cell conture.

Materials

• FBS
• Frozen HeLa cells in vials stored in liquid Nitrogen
• Complete medium with 10% FBS
• Water bath
• T-25 flask (CORNING)
• 15 ml centrifugal tubes.

Protocol

To prevent osmotic shock and maximize cell survival, use the following procedure to start a new culture from frozen cells.

1. Prepare about 1 L 37°C water, and about 10mL warm complete medium with 10% FBS in 15mL centrifuge tube.
2. Take frozen cells out from liquid nitrogen freezer and immediately place it into 37°C water, and keep shaking the cells to accelerate the melting process and try to thaw the cells within 60 sec.
3. Quickly transfer the thawed cell suspension into the prepared complete medium.
4. Centrifuge the cell suspension at 800 rpm at room temperature for 5 min and then discard supernatant.
5. Gently resuspend cell pellet in complete medium with 10% FBS and transfer the cells into properly labeled flask.
6. Culture the cells at 37°C in a humidified atmosphere of air and 5% CO2.
7. Check whether the cells have been attached to the plate after 12-24 hours.

Result

Observation

[2014 Aug 5th] Due to the pool condition of the long stored cell stock, most cell remain suspending in medium.