Biomedical Engineering Reference
In-Depth Information
a tube to deliver CO
2
that has come through a flow meter. We monitor CO
2
levels with a
separate portable CO
2
meter and run the system at steady state. In principle, one could
alternatively set up an integrated system with a CO
2
monitor that controls gas flow into the
air system. Humidity can be maintained by putting into the end of the warm air feed at the
enclosure a small reservoir for fluid with a paper towel wick. We suggest using Phosphate
buffered saline (PBS) instead of distilled water to prevent osmotic loading of water into the
dish of cells.
3.2.4 Observation Chambers
We routinely use sealed chambers that allow for uninterrupted
B
100 h film runs without
loss of cell viability or slowing of the cell cycle at later times for untransformed and
transformed human somatic cells. Film runs are typically terminated when the cultures
become confluent (
Figure 3.2
).
Details of chamber construction are described in Sluder et al.
[4]
. The only change we have
made in the assembly of the chambers is that we no longer introduce fluorocarbon oil into
the preparations.
Briefly, the chambers consist of a 1
3
3 inch aluminum slide 3 mm thick, with one or two
square cutouts (14 mm
3
14 mm) smaller than a 22 mm
3
22 mm coverslip for ample
overlap of the coverslip over the metal support to ensure a tight and durable seal (
Figure 3.3
).
We use only #1.5 (0.17 mm thick) coverslips for which all common objectives are
corrected. Use of any other thickness of coverslip produces spherical aberration that
substantially degrades image contrast and intensity. The thickness of the slide is intended to
(A)
(B)
Figure 3.2
Example of RPE1cell propagation at the start and end of a 136 h filming run. A and B are images of
the same field marked by the scribed circle. In this particular case, the medium was not changed
during the film run. Source: Preparation and images courtesy of Dr. Anna Krzywicka-Racka.
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