Because low cell numbers have been shown to be sufficient for IP-FCM analysis [6-8], we hypothesized that the multiplex approach could be applied to examine signalling-induced protein complexes from sr-T cells that were isolated from small clinical samples. Can the crawl-out method yield sufficient primary human sr-T cells for protein complex analysis using mIP-FCM? Identification of antibodies and description of the methods used for T cell harvesting and stimulation http://www.selleckchem.com/products/Y-27632.html are described in Data S1. The technical details and protocols of singleplex IP-FCM [6-8] and multiplex IP-FCM [5] have been previously published. Briefly, after incubation on ice for 20?min, lysates were centrifuged at 21?000?g for 10?min to discard nuclei. Multiplex immunoprecipitation was performed by adding to each postnuclear lysate, a mix of five specific antibody-conjugated Luminex beads. Following overnight incubation at 4��C, beads were washed on a magnetic plate washer (Bio-Rad, Hercules, CA, USA) and incubated with PE-conjugated probe antibodies, or with biotinylated probe antibodies followed by streptavidin-PE. After washing, beads were resuspended in IP-FCM buffer [150?mm NaCl, 50?mm Tris-HCl, 1% bovine serum albumin (Fraction-V, Fleroxacin Sigma-Aldrich, St. Louis, MO, USA)] and analysed on a Bioplex 200 multiplex bead reader. Duplicate mIP-FCM samples were acquired for all data associated with each donor. The median fluorescence intensity (MFI) of corresponding duplicate data was averaged, and pairwise protein complex data were expressed as the ratio of stimulated/unstimulated to indicate fold-change. CHIR-99021 mouse To statistically analyse data from both donors together, two-tailed Student's t-test was used to determine whether significant changes in protein complexes occurred in stimulated versus unstimulated conditions. Results are reported at both P?<?0.05 level, and the Bonferroni-adjusted level P?<?0.005 (based on 10 pairwise comparisons). The crawl-out method of cutaneous T cell isolation yielded on average 1.7?��?0.2?��?105 cells from the cytomatrix supports (mean?��?SEM). Cellular flow cytometry showed that greater than 90% of isolated cells were CD3+ (data not shown), with approximately 71% of T cells being CD4+ and 22% CD8+ (illustrated for one donor, Fig.?1a). Furthermore, approximately 90% of T cells were clearly CLA+CCR4+ (Fig.?1b). These data are similar to previous reports using the crawl-out method [3, 4], indicating that isolation of primary human sr-T cells was achieved as expected. Multiplex IP-FCM captures target proteins on Luminex microspheres and then detects co-associated proteins using fluorophore-conjugated probe antibodies (Fig.?1c). This allows flow cytometry-based semiquantitative analysis of proteins bound to each other in shared complexes.</div>
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