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
Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction.
Authors:Lin P, Correa D, Lin Y, Caplan AI,
Journal:PLoS One
PubMed ID:21887340
'Human mesenchymal stem cells (hMSCs) can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the ... More
Identifying disruptors of male germ cell development by small molecule screening in ex vivo gonad cultures.
Authors:Wakeling SI, Miles DC, Western PS,
Journal:BMC Res Notes
PubMed ID:23631647
'Germ cell development involves formation of the spermatogenic or oogenic lineages from the bipotential primordial germ cells. Signaling mechanisms in the fetal testis and ovary determine whether germ cells enter the male or female developmental pathway, respectively. These signaling processes underpin an important phase of germ cell development, disruption of ... More
Constitutive phosphorylation of GATA-1 at serine²6 attenuates the colony-forming activity of erythrocyte-committed progenitors.
Authors:Lin KR, Li CL, Yen JJ, Yang-Yen HF,
Journal:
PubMed ID:23717580
We previously reported that IL-3 signaling induces phosphorylation of GATA-1 at the serine²6 position, which contributes to IL-3-mediated anti-apoptotic response. Here, we demonstrate that phosphorylation of GATA-1 at serine²6 is also transiently induced in cells of the erythroid lineage (primary erythroblasts and erythrocyte-committed progenitors [EPs]) by erythropoietin (EPO), the principal ... More
Aeroallergen challenge promotes dendritic cell proliferation in the airways.
Authors:Veres TZ, Voedisch S, Spies E, Valtonen J, Prenzler F, Braun A,
Journal:J Immunol
PubMed ID:23267021
Aeroallergen provocation induces the rapid accumulation of CD11c(+)MHC class II (MHC II)(+) dendritic cells (DCs) in the lungs, which is driven by an increased recruitment of blood-derived DC precursors. Recent data show, however, that well-differentiated DCs proliferate in situ in various tissues. This may also contribute to their allergen-induced expansion; ... More