title: Detection of intracellular ADAMTS13, a secreted zinc-metalloprotease, via flow cytometry.
abstract: ADAMTS13 is a secreted metalloprotease that cleaves von Willebrand Factor multimers in order to maintain proper homeostasis. A severe deficiency in ADAMTS13 triggers a disorder known as thrombotic thrombocytopenic purpura. At present, ADAMTS13 expression levels are determined by immunoblotting. We established a flow cytometry methodology to detect intracellular ADAMTS13 in liver and kidney cells using a polyclonal antibody, BL154G, and several monoclonal antibodies previously used to detect ADAMTS13 by immunoblotting. The results were validated using confocal microscopy, immunoblotting, and an activity assay (FRETS-VWF73). We show that labeling ADAMTS13 with specific antibodies and detection by flow cytometry yields results that are comparable with previously established methods for ADAMTS13 detection. Specifically, we compared the endogenous expression levels of ADAMTS13 in various liver cell lines using flow cytometry and obtained results that parallel immunoblot analysis. Knockdown of ADAMTS13 expression via targeted siRNA resulted in significantly reduced median signal, displaying the sensitivity of this detection method. A further analysis of reliability and specificity was achieved through plasmid DNA and transfection reagent dose response studies. The flow cytometry method described here is useful in determining the expression of both endogenous and recombinant forms of intracellular ADAMTS13. Flow cytometry is a convenient, efficient, and cost-effective way to measure the expression levels of ADAMTS13.
authors.affiliation: Division of Hematology, Laboratory of Hemostasis, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
authors.forename: S
authors.initials: S
authors.lastname: Geetha
authors.forename: Courtni E
authors.initials: CE
authors.lastname: Allen
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Hunt
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Plum
authors.forename: Susan
authors.initials: S
authors.lastname: Garfield
authors.forename: Scott L
authors.initials: SL
authors.lastname: Friedman
authors.forename: Kenji
authors.initials: K
authors.lastname: Soejima
authors.forename: Zuben E
authors.initials: ZE
authors.lastname: Sauna
authors.forename: Chava
authors.initials: C
authors.lastname: Kimchi-Sarfaty
mesh_terms: D051722:ADAM Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human