title: Synchrotron radiation X-ray micro-fluorescence: Protocol to study mesenchymal stem cells.
abstract: The micro-X-ray fluorescence by synchrotron radiation (μ-XRF) is a method to determine the composition of tissues without destroying the samples. However, this technique has never been used for the analysis of mesenchymal stem cells (MSC). This study compared different protocols for fixing, storing, preserving, and establishing the correct numbers of dental derived MSC submitted to μ-XRF analysis. Stem cells were obtained from human dental tissue. After cell expansion, and MACS isolation, the samples were fixed and the following quantities of cells 1 × 10(4) to 1 × 10(7) were divided in two groups: G1: fixed in 4% paraformaldehyde diluted in phosphate-buffered saline solution, and G2: fixed in 4% paraformaldehyde diluted in MilliQ water. The G1 cells showed precipitation of chemical components from the solution resulting in the formation of salt crystals while G2 cells were clear and almost transparent in the sample holder. With regards to cells concentration, the best results occurred when four droplets of 1 × 10(7) cells were analyzed. This work shows that to identify and study the distribution of trace elements in MSC by μ-XRF, the best protocol is fixation in 4% paraformaldehyde diluted with MilliQ water at 4°C and a concentration of four incremental droplets of 1 × 10(7) cells.
authors.affiliation: Department of Pediatric Dentistry, University of São Paulo, São Paulo, Brazil.
authors.forename: Karla Mayra
authors.initials: KM
authors.lastname: Rezende
authors.affiliation: Department of Pediatric Dentistry, University of São Paulo, São Paulo, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Bönecker
authors.affiliation: XRF Line, Brazilian Synchrotron Light Laboratory, Campinas, Brazil.
authors.forename: Carlos Alberto
authors.initials: CA
authors.lastname: Perez
authors.affiliation: Department of Oral Pathology, University of São Paulo, São Paulo, Brazil.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mantesso
mesh_terms: D002478:Cells, Cultured; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000737:chemistry* / Q000166:cytology*; D008670:Metals / Q000032:analysis / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D013052:Spectrometry, X-Ray Emission / Q000379:methods*; D017356:Synchrotrons
chemical_list: D008670:Metals
keywords: X-ray analysis; protocols; stem cells; synchrotron radiation; trace elements