title: Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label.
abstract: Thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) has recently been introduced as an alternative to 5-bromo-2-deoxyuridine (BrdU) for cell labeling and tracking. Incorporation of EdU into replicating DNA can be detected by azide-conjugated fluors (eg, Alexa-azide) through a Cu(i)-catalyzed click reaction between EdU's alkyne moiety and azide. While this cell labeling method has proven to be valuable for tracking transplanted stem cells in various tissues, we have found that some bone marrow cells could be stained by Alexa-azide in the absence of EdU label. In intact rat femoral bone marrow, ~3% of nucleated cells were false-positively stained, and in isolated bone marrow cells, ~13%. In contrast to true-positive stains, which localize in the nucleus, the false-positive stains were cytoplasmic. Furthermore, while true-positive staining requires Cu(i), false-positive staining does not. Reducing the click reaction time or reducing the Alexa-azide concentration failed to improve the distinction between true- and false-positive staining. Hematopoietic and mesenchymal stem cell markers CD34 and Stro-1 did not co-localize with the false-positively stained cells, and these cells' identity remains unknown.
authors.affiliation: Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California 94143-0738, USA.
authors.forename: Guiting
authors.initials: G
authors.lastname: Lin
authors.forename: Hongxiu
authors.initials: H
authors.lastname: Ning
authors.forename: Lia
authors.initials: L
authors.lastname: Banie
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Qiu
authors.forename: Haiyang
authors.initials: H
authors.lastname: Zhang
authors.forename: Tom F
authors.initials: TF
authors.lastname: Lue
authors.forename: Ching-Shwun
authors.initials: CS
authors.lastname: Lin
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000818:Animals; D000831:Animals, Newborn; D018952:Antigens, CD34 / Q000737:chemistry; D000954:Antigens, Surface / Q000737:chemistry; D001386:Azides / Q000737:chemistry*; D001854:Bone Marrow Cells / Q000737:chemistry* / Q000166:cytology; D002467:Cell Nucleus / Q000737:chemistry; D057930:Click Chemistry / Q000379:methods; D003593:Cytoplasm / Q000737:chemistry; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000737:chemistry; D005189:False Positive Reactions; D005269:Femur / Q000737:chemistry / Q000166:cytology; D005434:Flow Cytometry; D009930:Organic Chemicals / Q000737:chemistry; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors
chemical_list: C417664:Alexa594; D000480:Alkynes; D018952:Antigens, CD34; D000954:Antigens, Surface; D001386:Azides; D009930:Organic Chemicals; C527498:STRO-1 antigen, rat; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine