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This methodology provides a powerful tool for unraveling the cellular biology and …


Biology Articles » Cell biology » An efficient rapid system for profiling the cellular activities of molecular libraries » Materials and Methods

Materials and Methods
- An efficient rapid system for profiling the cellular activities of molecular libraries

 
Automated Cellular Assays. The assay portion of the ACP consists of the same control softwareas the automated tissue culture station, a noncontact 1,536-wellreagent dispenser (dispense range of 250 nl to 5 µl at5% coefficient of variation (CV), dispense time of <1 min/plateat 5 µl), a 1,536-pin transfer device (validated at 9nl and >10% CVs), room-temperature hotels, the same incubatorsas described above, and a Perkin–Elmer ViewLux plate reader.To request cells for an assay, the operator inputs the desiredfinal number of assay plates, cell density, and well volume.The automated tissue culture station calculates the correctnumber of daughter flasks for pooling (if the number of cellsrequired is unavailable, the cell propagation protocol is enacted).When daughter flasks have incubated for a defined amount oftime, they are pooled in a matrix fashion into several emptyrecipient flasks. An equal volume from each daughter cell flaskwill be deposited into each recipient flask. A sample from onepooled flask will be counted (only one sample is necessary,because all of the flasks have been adjusted to the same celldensity by the pooling process). All of the flasks will havetheir volume adjusted to the desired cell density for plating.This pooling function, although complex, obviates the requirementfor a common pooling container, thus decreasing the possibilityof contamination. The flasks are then moved to the cell dispenser,where cells are pulled from flasks and dispensed into 1,536-wellassay plates, typically 5 µl/well. The plates of cellsare placed into an incubator and the empty flasks discarded.For the assay described in this paper, compounds are introducedto the system in 1,536-well plates through the room-temperaturehotels. 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. Thecell requests are processed as described above. After cell plating,preplated compounds in single-dose or dose–response formatare transferred from the compound plates to the assay platesby using the 1,536 pintool. The assay plates are incubated inthe environmentally controlled (37°C, 95% humidity, 5% CO2)incubators for 48 h (a custom lid design minimizes edge effectsfrom evaporation and enables 5-µl cell-based assays tobe incubated for up to 5 days). After incubation, 5 µl/wellCell Titer Glo (Promega) is added with the reagent dispenser,incubated for 10 min at room temperature, and luminescence quantitatedwith the Perkin–Elmer ViewLux. A luminescence value lessthan the control value indicates a decrease in either the numberof 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.


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