shRNA gene silencing Human Neuroblastoma cells (SH-SY5Y) Connexin 43

The estimation of DNA methylation level heavily depends on the complete conversion of non-methylated DNA cytosines. It is crucial to ensure complete conversion of non-methylated cytosines in DNA. Therefore, it is important to incorporate controls for bisulfite reactions, as well as to pay attention to the appearance of cytosines in non-CpG sites after sequencing, which is an indicator of incomplete conversion.

The estimation of DNA methylation level heavily depends on the complete conversion of non-methylated DNA cytosines. It is crucial to ensure complete conversion of non-methylated cytosines in DNA. Therefore, it is important to incorporate controls for bisulfite reactions, as well as to pay attention to the appearance of cytosines in non-CpG sites after sequencing, which is an indicator of incomplete conversion.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been greatly used for studies on embryonic development and cell differentiation.iPSCs provide a stable source for either self-renewal or differentiation into suitable cells when cultured in a particular environment. Pluripotent cell culture was originally started by deriving cells from inner cell mass (ICM) from pre-implanted blastocysts, these were called embryonic stem cells. These cells after isolation can be grown on traditional extracellular matrices (like mouse embryonic fibroblasts, MEFs) or feeder-free culture systems. DMEM/F12 has been the most commonly used basal media in the culture of pluripotent cells. These cells are cultured at normal atmospheric oxygen levels, 21%, however, some studies have proposed that 4% oxygen tension may be better for hESC growth. Higher D-glucose concentration (4.2g/l) and osmolarity (320mOsm) that mimics the natural environment of embryonic tissue are optimal for the growth of hESCs. Supplements like N2 and/or B-27, in the presence of growth factors like bFGF, have been shown to increase pluripotency of these cells. bFGF, FGF2 and other ligands of receptor tyrosine kinases like IGF are also required or maintain self-renewal ability of these cells. TGF𝛃1, by its activation of SMAD2/3 signalling, also represses differentiation of iPSCs. Other compounds like ROCK inhibitors reduce blebbing and apoptosis in these cells to maintain their clonogenicity. However, an inhibitor for LIF (leukaemia inhibitory factor, which is one of the pluripotent genes) has an opposing effect. Therefore, it is important to understand the culture conditions and media composition that affect downstream signalling in hESCs or iPSCs that may lead to their differentiation.

Get tips on using PE anti-mouse CD49b (pan-NK cells) Antibody to perform Flow cytometry Anti-bodies Mouse - CD49b

Get tips on using MEBMTM Mammary Epithelial Cell Growth Basal Medium to perform 3D Cell Culture Media Human primary breast ephitelial cells-Mammospheres

Get tips on using In Situ Cell Death Detection Kit, TMR red to perform TUNEL assay cell type - HEK293 human embryonic kidney cells

Get tips on using DMEM/F-12, no phenol red to perform 3D Cell Culture Media Human breast cancer MCF-7 cells-Mammospheres

Get tips on using TACS® 2 TdT Fluorescein Kit to perform TUNEL assay cell type - A127, U87MG, U251MG, T98G human glioblastoma cells

Get tips on using ROS-ID® Total ROS detection kit to perform ROS assay cell type - human umbelical vein endothelial cells (HUVEC)