کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9012274 | 1124550 | 2005 | 10 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A high-throughput chemotaxis assay for pharmacological characterization of chemokine receptors: Utilization of U937 monocytic cells
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کلمات کلیدی
PBSCXCRCCR7ATCCPhytohaemagglutininPHAPVPCCRPBMCSite-directed mutagenesis - mutagenesis مواجه با سایتChemokine antagonist - آنتاگونیست کیموکینHuman - انسانPeripheral blood mononuclear cell - سلول تک هسته ای خون محیطیLeukocyte - لکوسیتAmerican Type Culture Collection - مجموعه فرهنگی نوع آمریکاییPhosphate-buffered saline - محلول نمک فسفات با خاصیت بافریCell migration - مهاجرت سلولیMethods - مواد و روش هاpolyvinylpyrrolidone - پلی وینیل پیرولیدونCXC chemokine receptor - گیرنده شیمیایی CXCCC chemokine receptor - گیرنده کیموکین CC
موضوعات مرتبط
علوم پزشکی و سلامت
داروسازی، سم شناسی و علوم دارویی
داروشناسی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Introduction: Higher-throughput chemotaxis assays have had limited use in chemokine receptor pharmacology studies mainly because of the unavailability of optimal assay formats in addition to an incompatibility of chemotactic cell backgrounds with other pharmacological assays. Here, we developed a high-throughput 96-well chemotaxis assay for leukocytic cell lines and identified the human U937 monocytic line as an excellent cell background for both chemotaxis and the high-throughput calcium mobilization Fluorescent Imaging Plate Reader (FLIPR) assay. Methods: Optimal chemotactic conditions were developed using the Neuroprobe MBA96 nondisposable and the Millipore MultiScreen-MIC disposable apparatuses with responses to CXC chemokine receptor (CXCR)-4 endogenously expressed on the human H9 T lymphoma line, and confirmed with Jurkat T cell and U937 monocytic cell lines. Results: The U937 cell line was chosen for site-directed mutagenesis studies with CC chemokine receptor (CCR)-7 because this cell line did not endogenously express this receptor, it demonstrated a good chemotaxis index, and it showed an exceptional ability to mobilize calcium measured via FLIPR. Using the Millipore MultiScreen-MIC and FLIPR assays, alanine substitutions at K130 and Q227 caused threefold shifts in potency for the CCR7 ligand, CCL19, whereas that at K137 had no effect. Discussion: Because these CCR7 mutations have previously been shown not to affect ligand binding, our results here show that these residues are specifically involved in receptor activation signals critical to chemotaxis and underscore the importance of using the U937 cell background to confirm results of chemotaxis with those of the FLIPR assay.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Pharmacological and Toxicological Methods - Volume 51, Issue 2, MarchâApril 2005, Pages 105-114
Journal: Journal of Pharmacological and Toxicological Methods - Volume 51, Issue 2, MarchâApril 2005, Pages 105-114
نویسندگان
Thomas R. Ott, Anil Pahuja, Francisco M. Lio, Monica S. Mistry, Molly Gross, Sarah C. Hudson, Warren S. Wade, Pedro B. Simpson, R. Scott Struthers, David G. Alleva,