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

Journal: Cancer Research

Article Title: A Novel Small Molecule Inhibitor of Signal Transducers and Activators of Transcription 3 Reverses Immune Tolerance in Malignant Glioma Patients

doi: 10.1158/0008-5472.can-07-1243

Figure Lengend Snippet: Figure 4. WP1066 enhances phosphorylation of intracellular signaling molecules. Monocytes and Tcells isolated from the PBMCs of GBM patients were incubated with either the medium or medium supplemented with 5 Amol/L WP1066. After 2 h, monocytes and T cells were stimulated for 5 min with 2 Ag/mL LPS and 5 Ag/mL anti-CD3 antibody, respectively. Subsequently, cells were lysed, electrophoretically fractionated in 8% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10. A, for monocyte membranes, subsequently the same membrane was stripped and reblotted with antibodies to phosphorylated p72Syk (Tyr352), Syk, phosphorylated Hck, Hck, and Lyn. B, for the T-cell membranes, after stripping, the membrane was reblotted with antibodies to phosphorylated ZAP-70 (Tyr319), ZAP-70, p56-Lck, and h-actin.

Article Snippet: T cells from the same patients were isolated with human CD3 microbeads and a magnetic separation column (LS MACS column, Miltenyi Biotec) with the exception of one patient who lacked sufficient PBMCs for both monocyte and T-cell isolations.

Techniques: Phospho-proteomics, Isolation, Incubation, Membrane, Stripping Membranes