Procedure A. Doubling Number (PD)
- PD Number and Split Ratio are used to track the age of the cell culture and should be recorded on the cultureware.
- The current PD Number of the cells and the Split Ratio used should be recorded at the time the cells enter the new cultureware.
- The current PD Number is calculated by adding to the previously recorded PD Number the number of population doublings it took for the culture to reach confluency. The number of population doublings occurring between the two split events is determined by the previously recorded Split Ratio. See Table 1 and Example 1.
Table 1 - Split Ratios and Doublings
||Doublings to Confluency**
|*Used for information purposes only.
|**Used in calculating new PD Number
If a confluent flask--previously split at 1:2--is labeled as follows:
and is to be split 1:4, then the new cultureware should be labeled as follows:
PD15 (i.e. 14+1)
Procedure B. Feeding of Cells
1. Prepare TC hood for use.
2. Prior to feeding, media can be warmed (20 to 37oC) in a water bath or warming pad.
3. Mist media bottles and cultureware with 70% ethanol and transfer into the TC hood.
4. Aspirate used medium with a sterile (autoclaved) aspirating pipet.
5. Feed the culture with appropriate medium volume, according to Table 2.
6. To avoid cross contamination between cell lines, do not feed different cell lines in the TC hood at the same time.
7. Cover/loosely cap the culture and return to incubator.
Table 2 - Feed Volumes According to Dish/Flask Size
||Relative Area (P35=index)
||Feed Volume (mL)
*Cultures more than 50% confluent may require more medium.
Cultures less than 50% confluent should be fed this volume of medium.
293 cells will grow in smaller media volumes.