Click-iT® EdU kitを用いて 細胞増殖アッセイを行っています。 どのポイントで途中停止をかけられますか? あるいは、全てのステップを連続して行う必要がありますか?
EdU はDNAに化学的に取り込まれています。 そのため、サンプルを固定後、PBSを加えて4℃で一晩保存することが可能です。また、クリック反応により、Alexa Fluor® azide はEdUに共有結合で結合します。 そのため、クリック反応後も、PBS中で 4℃で一晩保存することが可能です。
Click-iT Plus EdU Imaging Kit は、今までの Click-iT EdU Imaging Kit と何が異なるのですか?
Click-iT EdU Imaging Kit では、反応バッファーに含まれていた銅イオンによって、GFPやR-PEが消光するなど、他の検出系との互換性の問題がありました。 Click-iT Plus EdU Imaging Kit では、銅イオンにキレート剤を付加することにより他の物質への影響を減少させました。 それに伴い、今までの Alexa Fluor azide を Alexa Fluor picolyl azide という物質に変更することで、クリック反応そのものの効率が落ちないようにしています。 Click-iT Plus EdU Flow Kit につきましても同様です。
I will be performing a cell proliferation assay using Click-iT EdU kit. At what point can I stop overnight, or do I have to perform all the steps continuously?
One may store the sample after fixation overnight in PBS at 4oC. For longer storage (<1 week) , store in buffer with 1-2% formaldehyde or in formalin to limit microbial growth. If you use sodium azide as a microbial inhibitor, it must be completely removed prior to the Click-iT reaction.
Can I combine Click-iT or Click-iT Plus reactions with phalloidin conjugates used for actin staining?
We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper.
For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here.
Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining.
Are the Alexa Fluor azides from Click-iT EdU kits available separately?
Yes, but the standalone products are not shipped at the same amount as provided in the Click-iT EdU kits; the amount of dye-azide provided in the Click-iT kits is proprietary information. See these catalog numbers for the standalone products:
- Cat. No. A10266: Alexa Fluor 488 azide
- Cat. No. A10270: Alexa Fluor 594 azide
- Cat. No. A10277: Alexa Fluor 647 azide
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IDO1 metabolites activate ß-catenin signaling to promote cancer cell proliferation and colon tumorigenesis in mice.
Authors:Thaker AI, Rao MS, Bishnupuri KS, Kerr TA, Foster L, Marinshaw JM, Newberry RD, Stenson WF, Ciorba MA
Journal:
PubMed ID:23669411
Indoleamine 2,3 dioxygenase-1 (IDO1) catabolizes tryptophan along the kynurenine pathway. Although IDO1 is expressed in inflamed and neoplastic epithelial cells of the colon, its role in colon tumorigenesis is not well understood. We used genetic and pharmacologic approaches to manipulate IDO1 activity in mice with colitis-associated cancer and human colon ... More
Lung myofibroblasts are characterized by down-regulated cyclooxygenase-2 and its main metabolite, prostaglandin E2.
Authors:Gabasa M, Royo D, Molina-Molina M, Roca-Ferrer J, Pujols L, Picado C, Xaubet A, Pereda J
Journal:
PubMed ID:23755232
Prostaglandin E2 (PGE2), the main metabolite of cyclooxygenase (COX), is a well-known anti-fibrotic agent. Moreover, myofibroblasts expressing a-smooth muscle actin (a-SMA), fibroblast expansion and epithelial-mesenchymal transition (EMT) are critical to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our aim was to investigate the expression of COX-2 and PGE2 in human ... More
An organotypic coculture model supporting proliferation and differentiation of medullary thymic epithelial cells and promiscuous gene expression.
Understanding intrathymic T cell differentiation has been greatly aided by the development of various reductionist in vitro models that mimic certain steps/microenvironments of this complex process. Most models focused on the faithful in vitro restoration of T cell differentiation and selection. In contrast, suitable in vitro models emulating the developmental ... More
PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways.
Authors:Shirai H, Poetsch AR, Gunji A, Maeda D, Fujimori H, Fujihara H, Yoshida T, Ogino H, Masutani M,
Journal:
PubMed ID:23744356
Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different ... More