Plant Cell Lines in Cell Morphogenesis Research - Plant Cell Morphogenesis: Methods and Protocols - page 215

Fig. 2 Growth characteristics of a cell suspension culture. ( a ) Typical growth curve shows the multiplication
rate of cell culture during 7-day subculture interval. Error bars = SEs ( n = 10). Growth phases of cell culture
during subculture interval: lag-phase (day 0-1), exponential phase (day 2-5), stationary phase (day 6-7).
( b ) Typical distribution of cell lengths and cell diameters in 2-day-old cell population ( n = 700)

3. Add 0.2 l Hoechst dye from stock solution per 1 ml of cell

suspension to get 0.2

μ

μ g/ml concentration and leave shaking

for 10 min.
4. Observe cells under the fl uorescence microscope and count
the total number of cells and the number of cells in mitotic
phase (for mitotic phases, see ref. [ 18 ]). Count at least 1,000
cells per sample in several optical fi elds ( see Notes 19 and 20 ).
5. Calculate mitotic index as the number of cells in mitosis divided
by the total number of counted cells, and plot the graph as a
function of time (days after subcultivation, e.g., see ref. [ 19 ]).

Commonly determined characteristics are cell size, cell shape, and
morphology of cell fi les or aggregates ( see Note 21 ).
1. For observation of suspension-cultured cell lines, use upright
or inverted light microscope; the application of both fl uores-
cent and Nomarski DIC equipment is highly recommended.
2. For saving and further processing the image data, use ade-
quate camera connected to a computer equipped with image
analysis software (e.g., ImageJ and NIS-Elements).
3. Adjust the cell density by dilution of cell suspension so that
cells on the microscopic slide do not overlap.
4. The most suitable objectives for observation and capturing of
images for further determination of cell length and diameter
are 10×, 20× or 40×. Save a set of images of each variant.
5. Determine the length and diameter of the cells using measure-
ment tools in image analysis software. Assess the parameters of
at least 400 cells in a set ( see Note 22 ).

3.8 Characterization
of Micromorphology
of Cell Lines