QUANTITATION is required in cell culture for the

characterization of the growth properties of different cell

lines.

Counting technology:
• Hemocytometer using a slide chamber and a microscope.

2)Electronic counting by electrical resistance.

3)Stained monolayers Crystal violet, Coomasie blue,

sulforhodomine B, MTT and measure the absorbance value.
 
QUANTIFICATION OF CELL PROLIFERATION
 
Cell weight ie 2.5 x108 cells /gram HeLa cells (14-16um in
diameter)
DNA content; assay DNA uses fluorescence method. Read
fluorescence emission at 492nm.
Protein; total cellular material can be measure by absorbance
of 280nm and colourmetric assays (Bradford reaction,
coomassie blue).
Rates of synthesis; DNA synthesis or RNA synthesis
•Estimation of DNA synthesis by (3H)Thymidine
incorporation
•Estimation of protein synthesis by (3H)leucine or (35S)
methionine and measure with scintillation counting
Enzymes assays and immunoassay;

Cytometry:

•in situ labeling; uses fluorescence dye or conjugated

antibody to detect antigen can measure amounts of
enzymes, DNA, RNA protein, and other constituents in
situ using CCD camera.

•flow cytometry; of a suspension cells able to measure

multiples constituents and activities
CELL proliferation

Measurement of cell proliferation rates are used to

set conditions for routine maintenance (subculture,
optimum dilution, estimate plating efficiency) or to
assay differences in growth maintenance (medium,
serum, culture flask etc).
Growth cycle parameters:

•Lag phase (cell adaptation and attaches to substrate, increase

enzymes activity follow by synthesis of new DNA and structural
proteins)
•Log phase (exponential growth (90-100%): consistent, uniform and
highest yield and reproducibility)
•Stationary Phase (plateau: reduce growth fraction (0-10%),
different morphology, more differentiated, secrete more
extracellular matrix and difficult to disaggregate), cell
concentration cells/ml medium in plateau.
•Saturation density
•Duration of Population doubling time (PTD). Time taken for the
culture to increase twofold in the middle of the exponential phase. ie
rapidly growing cells PTD of 12-24hr, slow growing cells PTD >24
hour.
Growth Curve with a monolayers in
flask

Growth Curve with suspension cultures

Growth Curve
Saturation Density
Plating efficiency

Define as colony formation at low density.

Prefer method for analyzing cell proliferation and survival.

Plating efficiency=No.of colonies formed/no.of

cells seeded x100

Seeding efficiency=No. of cells attached/no. of

cells seeded x100
Labelling Index

Cells that are synthesizing DNA will incorporate (3H)TdRIs.

Labelling Index is a percent of labeled cells as determined by

autoradiography. Able to show differences between
exponential growing cells and cells in plateau phase.

Growth Fraction; proportion of cells in cycle at time of

labeling.
Mitotic Index; percentage of cells in mitosis following staining
the cultures
Division index; percentage of cells in division cycle. Uses
antibodies directed against DNA polymerase and the
proportion of stained cells are determined by flow cytometry.
More sensitive than DNA labeling and mitotic index.