B. Novotn\'a et al. (dec 2019)
Journal of medicinal chemistry 62 23 10676--10690
Enzymatic Preparation of 2'-5',3'-5'-Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.
Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-$\kappa$B (I$\kappa$B) kinase (IKK)/nuclear factor-$\kappa$B (NF$\kappa$B) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.
RosetteSep™ Human CD8 Depletion Cocktail
RosetteSep™ Human Monocyte Depletion Cocktail
EasySep™ Release Mouse PE Positive Selection Kit
EasySep™ Mouse PE Positive Selection Kit II
Pearce DJ and Bonnet D (SEP 2007)
Experimental hematology 35 9 1437--46
The combined use of Hoechst efflux ability and aldehyde dehydrogenase activity to identify murine and human hematopoietic stem cells.
OBJECTIVE: In murine hematopoietic tissue, direct repopulation experiments have demonstrated that the side population (SP) represents a remarkable enrichment of hematopoietic stem cells. Human SP has been phenotyped as negative for lineage antigens as well as CD34. However, in the 9 years since the original publication, no long-term hematopoietic reconstitution has been reported for the adult human SP/CD34(-) subset. Elevated levels of aldehyde dehydrogenase (ALDH) have been demonstrated in murine and human progenitor cells when compared to other hematopoietic cells. METHODS: Here, we report the phenotype of human cord blood SP cells. We established the technique of simultaneous phenotyping, Hoechst exclusion, and ALDH labeling on murine tissues. We then performed the simultaneous analysis of phenotype, SP, and ALDH activity on human cord blood and bone marrow cells. Finally, we analyzed the phenotype and functional potential of human cord blood ALDH(+) cells to determine whether Lin(-)/CD34(-) cells are identified via this technique. RESULTS: We demonstrate that human Lin(-)/CD34(-)/ALDH(+) cells are capable of long-term repopulation. Although the SP technique identifies cells that overlap with the ALDH(+) cell population, this is restricted to the CD34(+) cell subset. CONCLUSION: Hoechst exclusion ability does not seem to be the method of choice for the isolation of human hematopoietic stem cells.