Protein expression and purification Insect cells Sf9

Get tips on using Quick-RNA Microprep Kit to perform RNA isolation / purification Cells - primary human aortic endothelial cells

Get tips on using NucleoBond® RNA/DNA to perform DNA isolation / purification Cells - Primary cells Rat cortical neurons

Get tips on using DNeasy Blood & Tissue Kit to perform DNA isolation / purification Cells - Primary cells Rat cortical neurons

Get tips on using Quick-RNA™ MiniPrep Plus to perform RNA isolation / purification Cells - primary human epithelial cells

Get tips on using APC Rat Anti-Mouse Ly-6G and Ly-6C to perform Flow cytometry Anti-bodies Mouse - Ly6C/Gr-1/Ly6G

Get tips on using Dead Cell Apoptosis Kit with Annexin V FITC and PI to perform Apoptosis assay cell type - OECM-1

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

Get tips on using Corning™ Basal Cell Culture Liquid Media - DMEM and Ham's F-12, 50/50 Mix to perform Mammalian cell culture media HSG cells

Get tips on using RIPA Buffer to perform Protein isolation Mammalian cells - SK-N-BE(2)-C

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.