Journal: Scientific Reports

Article Title: Ocimum flavone Orientin as a countermeasure for thrombocytopenia

doi: 10.1038/s41598-018-23419-x

Figure Lengend Snippet: Effect of Orientin on human hematopoietic stem cells. ( a – c ) Orientin induces human CD34 + cells differentiation towards MEP lineage. CD34 + cells purified from healthy donor’s PBMCs via density gradient ficol followed by magnetic bead separation were treated with or without Orientin in a serum free expansion medium (SFEM) supplemented with 100 ng/ml cytokine cocktail (CC100) for 7 days. Cells were harvested for lineage differentiation and stained with CD34-PE-Cy7, CD38-Pacblue, CD10-FITC, CD135-PE, CD45RA-APC antibodies and subjected to FACS analysis. (d) Orientin partially restores CD34 + cells differentiation potential after irradiation. CD34 + cells purified from healthy donor’s PBMCs (1 × 10 5 /ml in triplicates) were treated with or without Orientin (5 µM) and exposed to 4 Gy (fractionated dose of 2 × 2 Gy). These cells were then co-cultured on human mesenchymal stromal cell layer in SFEM medium supplemented with 100 ng/ml cytokine cocktail for normal differentiation in 7 day time period. The MEP cells were gated in lin − CD34 + CD38 + CD10 − CD135 − CD45RA − block in each sample. ( e ) Colony Assay in CD34 + cells (1 × 10 3 /ml in triplicates) post 0 or 2 Gy X-ray exposure. CFU-GM and GEMM colonies were scored on day 14 by applying standard morphologic criteria.

Article Snippet: CD34 + cells were purified from PBMCs using Diamond CD34 isolation kit for human (#130-094-531, MiltenyiBiotec) as described by manufacturer.

Techniques: Purification, Staining, Irradiation, Cell Culture, Blocking Assay, Colony Assay