Automated Cellular Assays.
The assay portion of the ACP consists of the same control software
as the automated tissue culture station, a noncontact 1,536-well
reagent dispenser (dispense range of 250 nl to 5 µl at
5% coefficient of variation (CV), dispense time of <1 min/plate
at 5 µl), a 1,536-pin transfer device (validated at 9
nl and >10% CVs), room-temperature hotels, the same incubators
as described above, and a Perkin–Elmer ViewLux plate reader.
To request cells for an assay, the operator inputs the desired
final number of assay plates, cell density, and well volume.
The automated tissue culture station calculates the correct
number of daughter flasks for pooling (if the number of cells
required is unavailable, the cell propagation protocol is enacted).
When daughter flasks have incubated for a defined amount of
time, they are pooled in a matrix fashion into several empty
recipient flasks. An equal volume from each daughter cell flask
will be deposited into each recipient flask. A sample from one
pooled flask will be counted (only one sample is necessary,
because all of the flasks have been adjusted to the same cell
density by the pooling process). All of the flasks will have
their volume adjusted to the desired cell density for plating.
This pooling function, although complex, obviates the requirement
for a common pooling container, thus decreasing the possibility
of contamination. The flasks are then moved to the cell dispenser,
where cells are pulled from flasks and dispensed into 1,536-well
assay plates, typically 5 µl/well. The plates of cells
are placed into an incubator and the empty flasks discarded.
For the assay described in this paper, compounds are introduced
to the system in 1,536-well plates through the room-temperature
hotels. Cell plates are requested at 105
cells/ml, 5 µl/well,
in 1,536-well plates, for each of the 35 Ba/F3 cell lines. The
cell requests are processed as described above. After cell plating,
preplated compounds in single-dose or dose–response format
are transferred from the compound plates to the assay plates
by using the 1,536 pintool. The assay plates are incubated in
the environmentally controlled (37°C, 95% humidity, 5% CO2
incubators for 48 h (a custom lid design minimizes edge effects
from evaporation and enables 5-µl cell-based assays to
be incubated for up to 5 days). After incubation, 5 µl/well
Cell Titer Glo (Promega) is added with the reagent dispenser,
incubated for 10 min at room temperature, and luminescence quantitated
with the Perkin–Elmer ViewLux. A luminescence value less
than the control value indicates a decrease in either the number
of cells or cell viability.
Supporting Text. Protocols detailing the automatic propagation of cell linesusing the ACP, as well as the construction of the Ba/F3/Tel-kinaselibrary, are presented in Supporting Text, which is publishedas supporting information on the PNAS web site.