Assay Development

Fluorescent Assays

Fluorescence can be used to measure; gene expression, protein stability, protein localization, organelle properties or cell homeostasis. We can measure fluorescence using either our high content microscope or with our plate reader.

Using microscopy, differences in can be processed and studied using MetaXpress and AcuityXpress. We currently have filters sets for DAPI/Hoechst, FITC/GFP, CY3/Deep Red, Texus Red and CY5/Far Red acquisition, so a maximum of five labels can be studied at once. Assay test plates and the library are arrayed on Perkin Elmer clear bottomed plates for all high content microscopy experiments (#60017460).

More rapid acquisition of fluorescence is done with the lab's plate reader, where the technique of in cell Westerns can be applied to gather total fluorescent levels. This type of analysis is excellent for robust screens ie, GFP-based transcriptional reporters or screens using an antibody recognizing a specific epitope. Plate reader dependent assays are usually performed where only quantitative data is needed. We use Matrix black, cell culture ready plates (#4318) for all GFP plate reader assays.

High Content Microscopy

The SRSF uses a Molecular Devices ImageXpress Micro to acquire images in 384 well format. We currently use black Perkin Elmer View Plates (#6007460), with 0.17mm plastic "coverslip" bottoms. Our library is pre-aliquotted in this plate type with 250ng of dsRNA per well. The layout in this plate type is the same as the other plates. The ImageXpress Micro has the filters sets for DAPI/Hoechst, FITC/GFP, CY3/Deep Red, Texus Red and CY5/Far Red acquisition, but if you need any other filter type, then you should contact us for further information. Imaging times depend upon a number of factors and you should also be prepared to spend some time developing the analysis methods.

Parameters that affect speed of acquisition are, images per well, numbers of filters used per screen, type of focusing and spacing of fields selected, all of which add only microseconds to each process, but over 53 plates of 384 wells amplify times. Screeners should also be aware a library screen generates between 0.5 and 2Terabytes of data. Images are acquired and raw images stored in our RNAi image server, capable of storing tens of Terabytes of data (the server is constantly growing).

The Metadata that is married with the images is stored on the MDCstore database housed within a solid-state drive housing the Metaserver. The images are processed using 4 dual bootable iMac workstations (quad-core) running Windows XP over a Gigabyte network. The images are processed using MetaXpress 3.1, derived from the MetaMorph software. Processing can be done in 2 ways. Images can either be segmented using predefined algorithms, or by writing an algorithm (known as a journal). Our predefined algorithms are capable of generating and measuring many different parameters from each image and detailed discussions with us are needed before embarking on an imaging screen.

The AcuityXpress software, which analyses the segmented images, can satisfy all your statistical needs and generate multiparametric analysis methods without the use of user generated spreadsheets.

Image Aquisition & Analysis
Image acquisition

Luminometry

We currently use a few different types of assays utilising firefly and renilla luciferases. However, the SRSF is not limited to these assays. The assays we use can be separated into transcriptional output, cell viability and b-galactosidase activity (as a function of the conversion of a luminescent assay reagent for quantitation of b-galactosidase in cells). For the transcriptional output, constructs are made employing a transcription factor binding sequence or enhancer, coupled to a minimal promoter driving firefly luciferase.

Cell number in this type of assay can be measured by co-transfection with a constitutively active promoter (Actin5C promoter) driving renilla luciferase, and compared as a ratio with the firefly luciferase reading. This ratio corrects for RNAi probes that alter cell viability or growth, which would otherwise be identified as "hits". The next type of luciferase assay we employ measures cell viability, by the release of ATP through the lysis of living cells. ATP concentration in the cell lysate is proportional to the number of cells present in culture and is the cofactor for luciferase activity and therefore can be used to measure total number of living cells.

Another screen assay has been developed to use luminescence as a reporter of b-galactosidase activity. This is particularly flexible as you can use b-galactosidase activity as a read-out for transcriptional activity and in protein complementation assays. For all assays using luciferase, including the library and assay test plates we use white cell culture ready Greiner Bio One Ltd (#781073) 384 plates.

Luminometry