eBook: Optimizing Cell Culture Techniques - 16

500 cells per microcavity. The plate was incubated
for 1 day at 37 °C, 5 % CO2
in a humidified incubator
to allow spheroid formation.
Experimental results (Corning) showed low
variability in spheroid size in each well across
the entire microplate, thanks to the accurate
dispensing of the cell suspension with the VIAFLO
96 (Figure 1 and 2).
Tips:
* The Z-height should be defined to provide
more accurate results
* Adjust the handle sensitivity as required by the
user for optimal automation of the process
2. Media exchange and washing steps
Changing cell culture media and washing the cells
without disturbing the spheroids
The VIAFLO 96 handheld electronic pipette was
used to pre-wet the plate with 50 µl of culture
media, which was then centrifuged at 500 x g for 1
minute to remove trapped air prior to cell seeding.
Three different cell lines (HT-29/GFP, HepG2 and
A549) were seeded using 100 µl per well at seeding
densities of 125, 250, 500 and 1000 cells per
microcavity (column per density). The plate was
incubated in a 37 °C, 5 % CO2
for 1 day.
humidified incubator
The next day, the VIAFLO 96 was used to add 20 µl
of Hoechst 34580 to the plate wells to stain the cell
nuclei, followed by incubation in a 37 °C, 5 % CO2
humidified incubator for 30 minutes.
The plate was then washed using the VIAFLO
96. The culture medium was removed, leaving
a volume of 50 µl per well, and 150 µl per well of
DPBS without calcium and magnesium was added.
Figure 4. Four washes with the VIAFLO 96 handheld electronic pipette
resulted in less than 5 % spheroid loss from 96 well Corning
Elplasia plates. The HT-29/GFP, HepG2, and A549 cell lines were
seeded at 125 to 1000 cells per microcavity and incubated overnight.
A VIAFLO 96 was used to add Hoechst stain to each well
and to perform 4 wash steps, removing all but 50 µl/well and
then adding 150 µl/well of DPBS. Each plate was imaged using a
CellInsight CX7 to count the number of spheroids per well before
and after each wash. Data shown with standard error of the
mean from three independent studies. N=24 wells.
Figure 3. Three cell lines formed consistent, single spheroids in
each microcavity of a 96 well Corning Elplasia plate. Representative
images of nuclei-stained HT-29/GFP, HepG2, and A549 cells
seeded at 500 cells per microcavity in a 96 well Corning Elplasia
plate. Images were taken with a CellInsight CX7 confocal imager
using a 4x objective with one field digitally zoomed in.
Finally, the plate was washed 4 times and imaged
after each wash to monitor spheroid loss.
16
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