350
Related Techniques
Subprotocol 2
Population Doubling Time
Population doubling time (PDT) is measured with a growing cell population during the exponential phase. Usually, when cells are seeded, the
cells multiply after a certain lag phase, and cease their multiplication
when they reach a certain cell density (plateau phase). During the phase
of active cell multiplication, cell numbers increase as 2n, where n is number of the cell generation. This phase of active cell multiplication is called
the exponential phase.
Materials
Equipment - Centrifuge and centrifuge tubes
- Hemocytometer
- Culture vessels
Solutions - Physiological saline
- Culture medium
Procedure
Cell culture 1. Harvest cells from the culture to be examined by suspending the cells
in the culture medium, and centrifuging the suspension at 150 g for 5
min.
Enumeration
of cells
Calculation
2. Wash the cells with physiological saline.
3. Suspend the cells in culture medium.
4. Seed the cells at an appropriate cell density (e.g., 5 x 10 5 cells/ml). The
cell density can be adjusted by measuring cell density with a hemocytometer, and by diluting the cell suspension with culture medium. If
the cell density of the suspension is below 5 x 10 5 cells/ml centrifuge
the suspension at 150 g for 5 min, and suspend the sedimented cells in
the volume of culture medium which resulted in 5 x 10 5 cells/ml cell
density.
1. Count the cells daily, and construct a growth curve to determine the
range of the exponential phase.
1. The PDT can be calculated from any two cell counts within the exponential phase using the following equation:
PDT = (t - to)10g2/(log N -log No)
where to is the time (hours or days) at which the first cell count was
made, t is the time arbitrarily set for the second cell count, and No and
N are the numbers of cells (per ml or per cm 2 ) at to and t, respectively.
Related Techniques
Subprotocol 2
Population Doubling Time
Population doubling time (PDT) is measured with a growing cell population during the exponential phase. Usually, when cells are seeded, the
cells multiply after a certain lag phase, and cease their multiplication
when they reach a certain cell density (plateau phase). During the phase
of active cell multiplication, cell numbers increase as 2n, where n is number of the cell generation. This phase of active cell multiplication is called
the exponential phase.
Materials
Equipment - Centrifuge and centrifuge tubes
- Hemocytometer
- Culture vessels
Solutions - Physiological saline
- Culture medium
Procedure
Cell culture 1. Harvest cells from the culture to be examined by suspending the cells
in the culture medium, and centrifuging the suspension at 150 g for 5
min.
Enumeration
of cells
Calculation
2. Wash the cells with physiological saline.
3. Suspend the cells in culture medium.
4. Seed the cells at an appropriate cell density (e.g., 5 x 10 5 cells/ml). The
cell density can be adjusted by measuring cell density with a hemocytometer, and by diluting the cell suspension with culture medium. If
the cell density of the suspension is below 5 x 10 5 cells/ml centrifuge
the suspension at 150 g for 5 min, and suspend the sedimented cells in
the volume of culture medium which resulted in 5 x 10 5 cells/ml cell
density.
1. Count the cells daily, and construct a growth curve to determine the
range of the exponential phase.
1. The PDT can be calculated from any two cell counts within the exponential phase using the following equation:
PDT = (t - to)10g2/(log N -log No)
where to is the time (hours or days) at which the first cell count was
made, t is the time arbitrarily set for the second cell count, and No and
N are the numbers of cells (per ml or per cm 2 ) at to and t, respectively.