BioProject

Basophil functional state was determined using the Flow2CAST Basophil Activation Test (BAT). More...

Basophil functional state was determined using the Flow2CAST Basophil Activation Test (BAT). Human basophils were isolated from whole blood of three house dust mite (HDM) responsive donors with reactive basophils and two donors with anergic basophils. Basophil isolation was performed using the EasySep Human Basophil Enrichment kit (Stemcell). Purity of the basophils was determined by flow cytometry and ranged from 96 - 99%. For stimulation, 75,000 basophils were incubated for 4 hr with or without 50 ul of anti-FCERI antibody (Bühlmann Laboratories) in a total volume of 200 ul of Stimulation Buffer provided by the Flow2CAST kit. After incubation, the basophils were collected and re-suspended in TRIzol Reagent (Life Technologies). Total RNA was extracted using double extraction protocol using the guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen), followed by a Qiagen RNeasy Micro clean-up procedure. cDNA and ST-ssDNA were prepared, fragmented and labeled according to Nugen WT Ovation Pico RNA Amplification and WT Ovation Exon kit and Encore Biotin protocols. The labeled ssDNA was hybridized on the Affymetrix Human GeneChip 1.0 ST Arrays, which subsequently were processed and stained using GeneChip Fluidics Station 450. The stained GeneChip Arrays were scanned in the Microarray Facility at the Biopolis Shared Facility using a GeneChip Scanner 3000. Quality control for the Arrays was performed using the Affymetrix Expression Console Software. Overall design: Basophil functional state was determined by BAT. Purified basophils were stimulated with or without anti-FCERI monoclonal antibody. Global gene expression was performed using the Affymetrix Human GeneChip 1.0 ST Arrays. Less...