By Ella Palmer (auth.), Ella Palmer (eds.)
As a excessive throughput approach for examining gene functionality, cell-based microarrays have confirmed to be of significant significance, permitting excessive throughput research of over expression and knock down of proteins. In Cell-Based Microarrays: tools and Protocols, specialists within the box supply an up to the moment synopsis of cell-based microarrays and meticulous assurance of all elements of the array, together with rising expertise. starting with an in depth evaluate of the total topic quarter, the amount maintains with protocols for over-expression arrays and downstream useful assays, infectious affliction examine, expanding transfection efficiencies, in addition to the advance of cell-based array know-how by way of use of microfluidic picture cytometry for the research of small diagnostic samples with few cells. Written within the hugely profitable equipment in Molecular Biology™ sequence structure, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and notes on troubleshooting and fending off identified pitfalls. finished and state of the art, Cell-Based Microarrays: equipment and Protocols serves as a key source for molecular biologists, geneticists, immunologists, and chemists, and offers scientists with entry to establish a know-how that's actually excessive throughput for the practical research of proteins.
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Extra resources for Cell-Based Microarrays: Methods and Protocols
Store at 25◦ C. 5. 2,000 ng/ml Standard curve DNA: 30 μl 2 μg/ml stock DNA, 1,470 μl 1× TE buffer. Large-Scale Cell-Based Microarrays 31 6. 200 ng/ml Standard curve DNA: 100 μl 2,000 ng/ml stock DNA, 900 μl 1× TE buffer. 7. 20 ng/ml Standard curve DNA: 10 μl 2,000 ng/ml stock DNA, 990 μl 1× TE buffer. 8. 2 ng/ml Standard curve DNA: 1 μl 2,000 ng/ml stock DNA, 999 μl 1× TE buffer. 9. Internal control stock (ICS): 20 μl 50 μg/ml human fetal liver genomic DNA (Biochain, Hayward, California, USA), 480 μl 1× TE buffer.
Add 1×107 cells per array to a 50-ml tube, make up to 20 ml with culture medium and invert to mix. 9. The arrays were placed in a square dish. Carefully pour the HEK293T cells onto the array avoiding direct contact with 38 Palmer and Freeman the printed areas and incubate the dish at 37◦ C, 5% CO2 until the cells are confluent – about 40 h. 7. Fluorescent Assays, Cell Visualisation and Counting Positive Cell Fluorescence We used two apoptotic assays, but any fluorescent assays can be used on the arrays.
The arrays were then treated with transfection reagent and cells grown over the top of the array until confluent. Cells growing in the vicinity of the spots of packaged genes were shown to be transfected and the encoded protein over-expressed. Arrays E. 1007/978-1-61737-970-3_3, © Springer Science+Business Media, LLC 2011 27 28 Palmer and Freeman can then be examined for alterations in cellular function, as manifested in localised changes to the cells’ biochemistry or morphology. If the expression vector contains a ‘tag’, then the sub-cellular localisation of the protein can also be analysed (1, 2).