This green avian macrophage is in the
process of phagocytizing red fluorescent E. coli. Some bacteria are
already present in phagosomes in the macrophage cell.
Avian Macrophage Phagocytosis
Avian Macrophage EST Libraries
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Avian
Macrophage Microarray
Description/Method
Manuscript
Data
An avian macrophage microarray (AMM) was constructed from clones derived from the unique elements from the four avian macrophage EST libraries plus some additional clones generously donated to us from the ChickEST database. The make-up of these clones is 4,772 clones from our libraries and 134 clones from ChickEST. PCR amplification of the inserts of these 4,906 clones was performed using Invitrogen PCR Supermix with vector appropriate primers and 1 microliter of plasmid DNA as template. The PCR products were purified using Qiagen QIAquick 96-well PCR purification kits. Products were dried-down and resuspended in 3XSSC/0.01%SDS spotting buffer. Spotting onto glass slides using a GeneMachines OmniGrid Accent spotter. The clones were spotted in triplicate, giving 14,718 total spots per slide. The arrays were tested by staining with a Panomer fluorescently labeled random 9-mer available from Molecular probes. The image from that staining is available here. This test showed most of the spots to contain cDNA product. Further, the array's usefulness was tested by hybridizing Cy3 and Cy5 labeled cDNA, created from unstimulated macrophage or LPS stimulated (2 hour) macrophage RNA respectively, to the slide. The labeled cDNAs were created using 50 micrograms of RNA and the Perkin Elmer Micromax direct labeling kit. The arrays were hybridized at 55C for 18 hours in an Advalytix ArrayBooster hybridization station. After washing off unbound label, the slides were scanned in an Applied Precision ArrayWoRxe microarray scanner. A picture of a portion of this slide with these two cDNAs hybridized and also stained with a Panomer is available here. Once these quality control experiments were completed, experimentation with these slides were ready to be performed.