Plant Cell Lines in Cell Morphogenesis Research - Plant Cell Morphogenesis: Methods and Protocols - page 214

cell suspension aliquots under continuous shaking on an orbital
incubator (orbital diameter 30 mm) in darkness at 27 °C and
150 rpm (BY-2 and VBI-0) or 25 °C and 130 rpm (LE).

3. Remove 1 ml sample of the cell suspension to a test tube every
day for a week (from a new aliquot in case of BY-2 and LE) or
every second day for 2 weeks (from the fi rst aliquot the fi rst week
and from the second aliquot the second week in case of VBI-0).
4. Use the samples for the cell density determination ( see
Subheading 3.6 ), alternatively also for the cell viability
assessment ( see Subheading 3.5 ).

1. To determine the viability of cells using the trypan (Evans)
blue, add 100

3.5 Assessment
of Cell Viability l of trypan blue solution per 1 ml of cell sus-

pension in the test tube. Observe staining via standard light
microscope ( see Note 14 ) and count the number of viable cells
as the percentage of the whole amount. Count at least 400
cells per sample in several optical fi elds.
2. To determine the viability of cells using the FDA assay, mix
aliquot of freshly prepared FDA-working solution (1:1 [v/v])
with cell suspension on a microscopic slide and observe via
fl uorescence microscope (excitation 494 nm; emission
521 nm). Count fl uorescing and non-fl uorescing cells not
later than 30 s after addition of FDA ( see Note 15 ). Taking
image data and postprocessing is advised in this case.

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3.6 Cell Density
Determination

1. Dilute the cell culture in test tube with modifi ed MS medium
(BY-2 and LE) or V4 (VBI-0) (Fig. 2c ) to get the fi nal num-
ber of cells between 500 and 4,000 cells for LE and 300 and
900 cells for BY-2 or VBI-0 in each counted chamber ( see
Note 16 ).
2. For cell density counting, use Fuchs-Rosenthal hemocytometer.
Count viable cells in the whole chamber. For each cell suspen-
sion variant, count at least ten repetitions ( see Notes 17 and 18 ).
3. Evaluate the density of cells according to the formula provided
with the hemocytometer or derived from chamber dimen-
sions. The cell number is usually expressed per milliliter of cell
suspension. Plot the values in a graph (Fig. 2a ).

Inoculate the usual amount of stationary cell suspension into fresh
medium (as described in Subheading 3.1 )

3.7 Assessment
of Mitotic Index
of Suspension-
Cultured Cells

1. During the SBI remove 1 ml aliquot of the cell culture to a
test tube. When necessary, dilute for the appropriate density
( see Subheading 3.5 ).
2. Add 10 l of 10 % solution of Triton X-100 per 1 ml of cell

suspension to get 0.1 % fi nal concentration.

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