title: A comparative study of PKH67, DiI, and BrdU labeling techniques for tracing rat mesenchymal stem cells.
abstract: Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1-3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3 ± 1.6% PKH67+ and 98.4 ± 1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2 ± 4.6% 10 μM BrdU+ and 77.6 ± 4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91 ± 3.8% PKH67+, 90 ± 1.5% DiI+, 91 ± 0.8% 5 μM BrdU+, and 76.9 ± 0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.
authors.affiliation: Institute of Neurobiology, Slovak Academy of Sciences, Center of Excellence for Brain Research, Soltesovej 4-6, 04001, Kosice, Slovakia, nagyovam@saske.sk.
authors.forename: Miriam
authors.initials: M
authors.lastname: Nagyova
authors.forename: Lucia
authors.initials: L
authors.lastname: Slovinska
authors.forename: Juraj
authors.initials: J
authors.lastname: Blasko
authors.forename: Ivana
authors.initials: I
authors.lastname: Grulova
authors.forename: Maria
authors.initials: M
authors.lastname: Kuricova
authors.forename: Viera
authors.initials: V
authors.lastname: Cigankova
authors.forename: Denisa
authors.initials: D
authors.lastname: Harvanova
authors.forename: Dasa
authors.initials: D
authors.lastname: Cizkova
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D005434:Flow Cytometry; D007150:Immunohistochemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals / Q000378:metabolism; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D002232:Carbocyanines; D009930:Organic Chemicals; C451241:PKH67; C024286:3,3'-dioctadecylindocarbocyanine; D001973:Bromodeoxyuridine
references.citation: Vet Immunol Immunopathol. 1999 Jan 4;67(1):33-45
references.pmid: 9950352
references.citation: Cell Mol Biol (Noisy-le-grand). 1995;41 Suppl 1:S121-32
references.pmid: 8574140
references.citation: Cytometry A. 2008 Nov;73(11):1019-34
references.pmid: 18785636
references.citation: Immunol Invest. 2007;36(5-6):713-38
references.pmid: 18161526
references.citation: C R Acad Sci III. 1998 Nov;321(11):901-7
references.pmid: 9879469
references.citation: Neuropsychopharmacology. 2001 Dec;25(6):799-804
references.pmid: 11750174
references.citation: Cytometry. 1995 Oct 1;21(2):160-9
references.pmid: 8582236
references.citation: J Immunol Methods. 1992 Dec 8;156(2):179-89
references.pmid: 1474255
references.citation: Blood Rev. 2006 May;20(3):161-71
references.pmid: 16364518
references.citation: Blood. 2010 Jul 1;115(26):5347-54
references.pmid: 20215639
references.citation: Proc Natl Acad Sci U S A. 1999 Sep 14;96(19):10711-6
references.pmid: 10485891
references.citation: Brain Res Rev. 2007 Jan;53(1):198-214
references.pmid: 17020783
references.citation: Cancer Res. 2000 Aug 15;60(16):4446-52
references.pmid: 10969791
references.citation: J Neurotrauma. 2011 Sep;28(9):1951-61
references.pmid: 20822464
references.citation: J Immunol Methods. 1997 Jun 23;205(1):55-65
references.pmid: 9236915
references.citation: J Cell Biol. 1996 Oct;135(1):63-71
references.pmid: 8858163
references.citation: Libyan J Med. 2007 Dec 01;2(4):190-201
references.pmid: 21503244
references.citation: Exp Hematol. 1998 Jan;26(1):53-62
references.pmid: 9430514
references.citation: J Cell Biol. 1997 Mar 10;136(5):1109-21
references.pmid: 9060475
references.citation: Stem Cells. 2002;20(6):501-13
references.pmid: 12456958
references.citation: Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11879-83
references.pmid: 8524867
references.citation: Brain Res. 2008 Jun 18;1215:11-9
references.pmid: 18468584
references.citation: J Neurocytol. 1989 Jun;18(3):311-8
references.pmid: 2746304
references.citation: Nature. 1989 Jul 13;340(6229):167-8
references.pmid: 2662017
references.citation: Blood. 1997 Jul 15;90(2):658-68
references.pmid: 9226166
references.citation: Science. 1999 Apr 2;284(5411):143-7
references.pmid: 10102814
references.citation: Cell Mol Neurobiol. 2006 Oct-Nov;26(7-8):1167-80
references.pmid: 16897366
references.citation: Prog Brain Res. 2007;161:367-83
references.pmid: 17618991
references.citation: Am J Physiol Lung Cell Mol Physiol. 2001 Jan;280(1):L58-68
references.pmid: 11133495
references.citation: Morphologie. 2000 Jun;84(265):11-5
references.pmid: 11048293
references.citation: Anticancer Res. 1998 Nov-Dec;18(6A):4181-8
references.pmid: 9891465
references.citation: Nat Med. 1999 Mar;5(3):309-13
references.pmid: 10086387
references.citation: In Vitro Cell Dev Biol Anim. 2001 Nov-Dec;37(10):646-55
references.pmid: 11776970
references.citation: Cell Transplant. 2008;17(8):911-22
references.pmid: 19069634
references.citation: J Immunol Methods. 1998 May 1;214(1-2):175-86
references.pmid: 9692869
references.citation: Semin Thorac Cardiovasc Surg. 2008 Summer;20(2):131-7
references.pmid: 18707646
references.citation: J Biol Chem. 1998 Dec 11;273(50):33354-9
references.pmid: 9837910
references.citation: Di Yi Jun Yi Da Xue Xue Bao. 2005 Feb;25(2):184-6
references.pmid: 15699001
references.citation: Stem Cells Dev. 2013 Jan 15;22(2):340-4
references.pmid: 22913652