Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate this assay, to allow the direct screening of drugs targeting signaling pathways. We show here the sensitivity of this fully automated assay on human growth hormone (hGH)-driven JAK/STAT5-activated IM-9 cells, and we discuss the throughput of this system, which is compatible with medium-throughput drug screening. Because the kit works directly on whole blood samples, ex-vivo assays are also possible with this approach, which could allow for the screening of drugs under more physiological conditions.
Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo-rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H-2K ing that it is recruited upon immunization for its optimal TCR-peptide/MHC fit. Thus, a CDR3b motif generated by a process akin to ''convergent recombination'' accounts for a sizable fraction of the alloreactive anti-K b TCR repertoire.
Introduction: Phospho-epitopes are difficult to detect by flow cytometry, currently requiring dedicated techniques and buffers, a total work time of 2 to 4 hours, 37°C and ice incubations, and 4 to 8 wash steps. The reference methods also uses methanol which is toxic for the user and harmful for the other cell markers, what compromises some multi-parametric studies. The FoxP3 marker is incompatible with “harsh” P-epitopes detection methods, rendering even more challenging the studies of Tregs functions. Methods: Here, we used a new commercially available kit; named PerFix EXPOSE (Phospho Epitopes Exposure Kit; for Research Use Only) based on multiple innovations in the permeabilization, staining, and wash steps. It is devoid of methanol and supports staining of all common P-epitopes with one single procedure of about 1 hour. Since many extra-cellular markers can be combined together with the anti-phospho markers, we evaluated also various FoxP3 clones and conjugates, and tried some procedure optimisations. The best conditions were then used to analyse the ex-vivo functionality of FoxP3+ cells: The IL-2 signalling pathway especially. Results: PerFix EXPOSE allows for the simultaneous detection of some antigens that are otherwise incompatible, such as FOXP3 and p-STATs. An IL-2 dose-response curve could be easily generated directly from fresh whole blood that confirmed on normal donors a 10 to 100-fold difference in sensitivity to IL-2 between Tregs and other T cells.
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