搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Monoclonal antibodies (mAbs) have proven to be instrumental in the advancement of research,diagnostic,industrial vaccine,and therapeutic applications. The use of mAbs in laboratory protocols has been growing in an exponential fashion for the last four decades. Described herein are methods for the development of highly specific mAbs through traditional hybridoma fusion. For ultimate success,a series of simultaneously initiated protocols are to be undertaken with careful attention to cell health of both the myeloma fusion partner and immune splenocytes. Coordination and attention to detail will enable a researcher with basic tissue culture skills to generate mAbs from immunized rodents to a variety of antigens (including proteins,carbohydrates,DNA,and haptens) (see Note 1). Furthermore,in vivo and in vitro methods used for antigen sensitization of splenocytes prior to somatic fusion are described herein. View Publication

A rapid,ultrasensitive,and practical label-free impedimetric immunoassay for measuring trace levels of total polychlorinated biphenyls (PCBs) in insulating oil was developed. First,we developed a novel monoclonal antibody (RU6F9) for PCBs by using a designed immunogen and characterized its binding affinity for a commercial mixtures of PCBs and its main congeners. A micro comblike gold electrode was fabricated,and the antibody was covalently immobilized on the electrode through a self-assembled monolayer formed by dithiobis-N-succinimidyl propionate. The antigen-binding event on the surface of the functionalized electrode was determined as the change in charge transfer resistance by using electrochemical impedance spectroscopy. The resulting impedimetric immunoassay in aqueous solution achieved a wide determination range (0.01-10 μg/L) and a low detection limit (LOD) of 0.001 μg/L,which was 100-fold more sensitive than a conventional flow-based immunoassay for PCBs. By combining the impedimetric immunoassay with a cleanup procedure for insulating oil utilizing a multilayer cleanup column followed by DMSO partitioning,an LOD of 0.052 mg/kg-oil was achieved,which satisfied the Japanese regulation criterion of 0.5 mg/kg-oil. Finally,the immunoassay was employed to determine total PCB levels in actual used insulating oils (n = 33) sampled from a used transformer containing trace levels of PCBs,and the results agreed well with the Japanese official method (HRGC/HRMS). View Publication

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of textgreater10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb,and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells. View Publication

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes,smooth muscle cells (SMC),and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs,differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result,numerous strategies have been developed to derive CPCs from ESCs. In this protocol,differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs,ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus,CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs. View Publication