Correlation between microarray results and Ag-specific T cell inputs.

<p>A guide to print one 10×5 cellular microarray in one subunit of a 4-chamber slide (A). Raw fluorescence images and measured Absolute Fluorescence Intensity (AFI) of an array chamber loaded with 100×10³ (B), 50×10³ (C), 20×10³ (D) and 5×10³ (E) of Ag-specific HA306, NAp43 and IGRPp31-specific CD4 T cells. No MPp54-specific T cells were loaded for assaying. Green fluorescence images on the right of each panel represent Ag-specific cytokine production signals probed by AlexFluor555-conjugated streptavidin while red fluorescence images on the left of each panel represent non-specific signals probed by Cy5-conjugated anti-Rat IgG to facilitate the spot identification. Band-pass filters avoided spectral overlap. Only the green fluorescence was measured as AFI and proceeded for quantification. Correlations between “<b>−log[</b><b><i>p</i></b><b>]</b>” and corresponding numbers of HA306, NAp43 and IGRPp31 specific CD4 T cells are shown in panel (F). MPp54 data in (F) represents Ag non-specific signals revealed by corresponding chip assays. Correlations between “<b>−log[</b><b><i>p</i></b><b>]</b>” and IFNgamma production for HA306 (G), NAp43 (H) and IGRPp31 (I) are also shown to determine the concordance between two different assays (microarray assay and ELISA).