The doubling time is the time it takes your cells to double in number. It is useful to know the doubling time of your cells so that you can plate the appropriate number for transduction with a lentiviral library or construct.

1. Start with cells that have already been growing for a few weeks, rather than using cells that have just been thawed from a frozen state. To calculate the doubling time, trypsinize your cells as if you were going to split them.
1. Count them using a hemacytometer or cell counter, and keep track of the number that you replate onto the cell culture plates. The starting number of cells (at the beginning) is Xb.
1. Propagate the cells as you normally do, replacing media as necessary.
1. The next time they are ready to be split, trypsinize them as usual and count them again using a hemacytometer or cell counter. The number of cells at the end is referred to as Xe.

NOTE: The cells should be in the log phase of growth to calculate doubling time properly, so it is important to not let the cells become confluent.

To calculate the doubling time, use the following formula:

Doubling Time = [ T × ( ln2 ) ] / [ ln (Xe / Xb) ]
where T = Time in any units

#### Example

Let’s say that on Day 0, you count 2 × 106 cells. Three (3) days later, you count 16 × 106 cells.

Xb = 2 × 106
T = 3 days
Xe = 16 × 106

Doubling Time = [ 3 × (ln2) ] / [ ln(16,000,000 / 2,000,000) ]
= [ 3 × (0.69) ] / [ ln(8) ]
= 2.08 / 2.08 = 1 day