Human neural progenitor cells differentiated by growth factor withdrawal
Note: Since classic Cadherins can be protected from trypsin treatment in the presence of Ca2+, cells in monolayer cultures are harvested with 0.01% Trypsin in the presence of 1-5 mM CaCl2 at 37° C. Flow cytometry can be performed according to the standard procedures, except that all the cell staining and washing steps are performed in the presence of Ca2+ and Mg2+ (e.g. using FACS buffer: PBS containing 1 mM CaCl2, 1 mM MgCl2, 2% FBS and 0.02% sodium azide).
|Cadherin‑12 in BG01V Human Embryonic Stem Cells. Cadherin‑12 was detected in immersion fixed BG01V human embryonic stem cells differentiated into neurons using Rat Anti-Human Cadherin‑12 Monoclonal Antibody (Catalog # MAB2240) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rat IgG Secondary Antibody (red; Catalog # NL013) and counterstained with DAPI (blue). Specific staining was localized to cell membranes. View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips.|
The cadherin superfamily is a large family of membrane-associated glycoproteins that engage in homotypic, calcium-dependent, cell-cell adhesion events. The superfamily can be divided into at least four subfamilies based on its member’s extracellular (EC) regions and cytoplasmic domains (1, 2). These include classical cadherins, desmosomal cadherins, protocadherins, and cadherin-like molecules that contain a variable number of EC and transmembrane (TM) domains (1).
Cadherin‑12, also known as brain-cadherin and N-cadherin 2, is a 150 kDa classical cadherin. Classical family molecules are modular in their extracellular region, mediating calcium-dependent cell-cell adhesion through their five EC Ca++-binding repeats (2). Cadherin-12 can be further identified as a type II classical cadherin, due to the absence of a His-Ala-Val motif in its most N-terminal cadherin repeat (3). Human Cadherin-12 is synthesized as a 794 amino acid (aa) type I transmembrane preproprotein that contains a 23 aa signal peptide, a 31 aa prosequence, a 555 aa extracellular region, a 28 aa transmembrane segment, and a 157 aa cytoplasmic domain (4, 5). The five EC cadherin domains are approximately 110 aa in length and generate two beta -sheets that are oriented like bread in a sandwich. Human Cadherin-12 EC region is 96% aa identical to mouse Cadherin-12 EC region. Cadherin-12 is expressed specifically in CNS neurons. The bulk of its expression is postnatal, and it is proposed to be involved in synaptogenesis (4). As a classic cadherin, Cadherin-12 will form homodimers and promote intercellular adhesion with itself and, possibly, cadherins-8 and -14 (6).
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