Brilliant violet fluorochromes in simultaneous multicolor flow cytometry-fluorescence in situ hybridization measurement of monocyte subsets and telomere length in heart failure.
Conventional analytical methods to determine telomere length (TL) have been replaced by more precise and reproducible procedures, such as fluorescence in situ hybridization coupled with flow cytometry (flow-FISH). However, simultaneous measurement of TL and cell phenotype remains difficult. Relatively expensive and time-consuming cell-sorting purification is needed to counteract the loss, due to stringent FISH conditions, of prehybridization fluorescence by the organic fluorochromes conventionally used in the phenotyping step. Here, we sought to assess whether the newly developed Brilliant Violet (BV) dyes are valuable to specifically and simultaneously assess the distribution and telomere attrition of monocyte subsets circulating in the blood of a cohort of patients with heart failure. We performed flow-FISH on blood samples from 28 patients with heart failure. To differentiate among monocyte subsets, we used BV and conventional fluorochromes conjugated to antibodies against CD86, CD14, CD16, and CD15. We simultaneously assessed the TLs of the monocyte subsets with a telomere-specific peptide nucleic acid probe labeled with fluorescein isothiocyanate. The BV dyes completely tolerated the harsh conditions required for adequate DNA denaturation and simultaneously provided accurate identification of monocyte subpopulations and respective TLs. The presented protocol may be faster and less expensive than those used currently for purposes such as establishing associations among patient categories, disease progression, monocyte heterogeneity, and aging in the context of heart failure.
ICREC Research Program, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain.|Center of Regenerative Medicine in Barcelona, Barcelona, Spain.
Santiago
S
Roura
Flow Cytometry Facility, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain.
Marco A
MA
Fernández
Cardiology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.|Department of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain.
Elena
E
Elchinova
Flow Cytometry Facility, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain.
Iris
I
Teubel
Flow Cytometry Facility, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain.
Gerard
G
Requena
Cardiology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.
Roser
R
Cabanes
Cardiology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.|Department of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain.
Josep
J
Lupón
ICREC Research Program, Germans Trias i Pujol Health Science Research Institute, Badalona, Spain.|Cardiology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.|Department of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain.
Antoni
A
Bayes-Genis
D000368:Aged; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006333:Heart Failure / Q000473:pathology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000473:pathology*; D059505:Telomere Homeostasis
D005456:Fluorescent Dyes