Vacutainer blood tubes

Human bone marrow samples were obtained from healthy adults (male, age =21 + 2.6 years) who were undergoing fracture fixation involving the long bones. After providing the patient information sheet and explaining the patients on bone marrow collection, written informed consent was obtained and bone marrow was collected. This study was approved by the University of Malaya Medical Center Ethics Committee (reference no. 602.22). Human bone marrow was collected (N = 6) in sterile 3-ml BD Vacutainer blood tubes (K2 EDTA, BD, Franklin Lakes, NJ, USA) by orthopedic surgeons and was kept at 4 °C until isolation. The mononuclear cells were isolated using Ficoll density gradient method as described earlier (Krishnamurithy et al., 2014 (link)). Isolated cells were cultured and expanded until P3 in T-75 flasks. The isolated cells were characterized as MSCs through flow cytometry using cell surface markers (positive and negative markers) and its ability to undergo trilineage differentiation (adipogenic, chondrogenic, and osteogenic differentiation) was described earlier (Tan et al., 2013 (link)). MSCs isolated form three biological samples were used for gene expression analysis and three biological samples were used for imaging analysis.

According to standard protocols, the fasting venous blood samples were collected in BD Vacutainer^® blood tubes. An earlier publication provided a thorough explanation of the sample collection and biochemical analysis methods [43 (link)]. Chemical-clinical analyses were done as part of routine laboratory testing, in a central laboratory (the University of Bremen, Centre for Biomolecular Interactions Bremen-CBIB). Serum samples stored at −80 °C were used to detect levels of hs-CRP, (using either single or MULTI-SPOT^® Assay Systems, Meso Scale Discovery, Rockville, MD, USA).
Taking advantage of the qPCR array technology, we previously determined c-miRNA profiles in children and adolescents of the I.Family study [40 (link)]. Methods for miRNA extraction and screening from plasma samples have been previously published [35 (link),40 (link)]. Individual plasma samples were first tested for hemoglobin levels and hemolyzed samples were omitted from the analysis [35 (link)]. Different assays were performed in triplicate by using the miScript Primer Assays according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Relative miRNA levels were normalized using the endogenous spike-in Cel-miR-39 [35 (link)] employing the Data Assist v3.1 software package (Life Technologies, Thermo Fisher Scientific, Milan, Italy).

Whole blood collected in BD Vacutainer blood tubes (BD Biosciences, UK) was diluted 1:1 in RPMI1640 medium and separated by centrifugation on Histopaque. Peripheral blood mononuclear cells (PBMCs) were harvested from the interface, washed and counted.