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cheal tube. Be careful not to overanesthetize, as the mice will
succumb to respiratory depression.- Thawing procedures are stressful for the frozen cells and using
the good techniques and fast thawing at 37 °C ensure high
proportion of the cells survive the procedures. Dilute the fro-
zen cells with pre-warmed complete media and mix with slow
pipette up and down. - Flask size depends on the number of frozen cells present in the
cryovial and culture conditions vary based on cell type and
media used. Generally cells seeded at 10,000 cells/cm^2 , in 5%
CO 2 at 37 °C used for routine culture and passages. - Add 2 mL and 5 mL 0.25% trypsin-EDTA in 25cm^2 and
75 cm^2 flask, respectively. Monitor the cells in every 2 min that
they are being disassociated. Tap gently the flask to get maxi-
mum disassociation and recovery of cells. Do not put long
time cells in trypsin, which may cause reduced viability of the
cells. - Add 0.4% trypan blue stock solution and cell suspension in a
1.5 mL centrifuge tube (1:1). For example, add 100 μL trypan
blue in 100 μL cells and mix by pipetting and load 10 μL of
this cell on a hemocytometer. Examine and count the viable
cells under microscope with low magnification. Count the blue
or dead cells (dead cells take up trypan blue and became
stained) and total number of cells. Cells viability is calculated
using the formula, % of viable cells = [1-(Number dead
cells ÷ Number of total cells)] × 100. Number of cells per mL
of culture can be determined using the following formula,
number of viable cells × 10^4 × dilution factor = cells/mL of
culture. At least 95% cell viability should be obtained for
healthy log phase culture of cells. - Low cell counts can lead to prolonged sorting duration and
might affect the viability of cells. Too many cells can reduce the
purity and cause blockages. Samples should contain 1 × 106 to
2 × 107 cells/mL for better sorting results. Also, ensure that
cells are in single cell suspension. No cell clumps or aggregates
are in cells. To avoid the formation of cell clumps, always use
Ca/Mg++ free PBS. If cells have higher tendency to aggregate,
add anti-clump agents such as EDTA (1–5 mM) to the buffer
to prevent formation of aggregates. Add 20–50 μg/mL DNAse
I and 5 mM MgCl 2 .6H 2 O to avoid cell clumping due to cell
death. - For accurate identification of SP population of cells, control
groups are required, in which the ATP-binding cassette-
transporters responsible for Hoechst 33342 dye exclusion are
blocked with inhibitor treatment. Most commonly used ATP-
binding cassette-transporter inhibitors are verapamil and fumi-
Farhadul Islam et al.