Part VI: Freezing and viability staining of cells
Welcome to the last of a series of articles providing useful hints for culturing
animal cells. This article contains considerations for freezing and viability
staining of cells.
For some cell cultures, especially valuable ones, it is common practice
to maintain a two-tiered frozen cell bank: a master and a working cell bank.
The working cell bank comprises cells from one of the master bank samples,
which have been grown for several passages before storage. If and when future
cell samples are needed, they are taken from the working cell bank. The
master cell bank is used only when absolutely necessary. This ensures that
a stock of cells with a low passage number is maintained, and avoids genetic
variation within the culture.
1. Check that cells are healthy, not contaminated, and have the correct
2. Change the medium 24 h before freezing the cells.
Tip - Adherent and suspension cell cultures should not be at a high density
for freezing. We recommend freezing cells when they are in the logarithmic
3. Adherent cultures: harvest the cells by trypsinization, resuspend in
medium containing serum, centrifuge at 200 x g for 5 min, and then resuspend
cells in freezing medium (see Table 1) at a density of 35 x 106 cells/ml.
Suspension cultures: centrifuge the cells at 200 x g for 5 min, and resuspend
in freezing medium (see Table 1) at a density of 510 x 106 cells/ml.
Freezing medium containing DMSO is hazardous and should
be handled with caution.
4. Transfer 1 ml of the cell suspension (approximately 35 x 106 adherent
cells or 510 x 106 suspension cells) into each freezing vial. Label
vials with the name of cell line, date, passage number, and growth medium.
Tip - It may also be useful to note the cell density in the freezing vials
before storing. This enables determination of the cell density that provides
optimal recovery after thawing.
5. Place freezing vials in racks and transfer to a polystyrene box (with
walls approximately 15 mm thick) lined with cotton wool. Store box in a
80C freezer overnight.
Tip - It is important that cells are frozen at a rate of 1C/min. A
controlled-rate freezing device can be used instead of the polystyrene box
and cotton wool method.
6. The next day, quickly transfer the vials to a liquid nitrogen chamber,
making sure that the vials do not begin to thaw.
Trypan blue staining provides a method for distinguishing between viable
(i.e., capable of growth) and nonviable cells in a culture. This staining
method is based on dye exclusion: cells with intact membranes
exclude (i.e., do not take up) the dye and are considered viable.
1. Harvest the cells, either by trypsinization (adherent cell cultures)
or by centrifugation at 200 x g for 5 min (suspension cell cultures). Resuspend
the cells in an appropriate volume of pre-warmed growth medium to give a
cell density of at least 106 cells/ml.
2. Add 0.5 ml 0.4% (w/v) trypan blue (see Table 1) and 0.3 ml PBS (see Table
1) or Hanks balanced salt solution (HBSS; see QIAGEN News 2002 No.
3, 22 for composition) to 0.1 ml of the cell suspension. Mix
and let stand for 12 min.
Tip - Alternatively, add 0.4 ml trypan blue directly to 0.4 ml of cells
in growth medium.
Tip - At least 106 cells/ml are required for accurate counting.
3. Count the stained and unstained cells using a hemocytometer (see QIAGEN
News 2002 No. 4, 28). Blue-stained cells are nonviable and unstained cells
No. of viable cells/ Total no. of cells = % viability
Table 1. Composition of solutions for animal cell culture protocols
These protocols have been adapted from the following references. Protocols
in this series are examples of methods for general cell culture and have
not been rigorously validated and optimized by QIAGEN. There are many alternative
protocols in current use.
1. Freshney, R.I. (1993)
Culture of Animal Cells:
a Manual of Basic
Technique. 3rd ed. New
2. Ausubel, F.M. et al., eds.
(1991) Current Protocols
in Molecular Biology.
New York: Wiley
3. Spector, D., Goldman,
R.R., and Leinwand, L.A.,
eds. (1998) Cells: a
Laboratory Manual. Cold
Spring Harbor, NY: Cold
Spring Harbor Laboratory
Source:Page: All 1 2 3 Related biology technology :1
. The QIAGEN Guide to Animal Cell Culture2
. The QIAGEN Guide to Animal Cell Culture3
. The QIAGEN Guide to Animal Cell Culture4
. The QIAGEN Guide to Animal Cell Culture5
. The QIAGEN Guide to Animal Cell Culture6
. QIAGEN Instrument Service insist on the best in service and support7
. QIAGEN Multiplex PCR Handbook8
. QIAGEN Multiplex PCR Kit9
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