固定・透過処理したサンプルでアポトーシスの確認をしたい。 どの試薬がおすすめか? 蛍光標識 Annexin V は使用できるか?
固定後のサンプルでの Annexin V の使用はお勧めできません。 ホルムアルデヒドによる架橋により、ホスファチジルセリンもマスクされる可能性があるためです。 固定後のサンプルで使用できるアポトーシス検出方法は、anti-Caspase抗体を用いた免疫染色、もしくは、 Click-iT® TUNEL Imaging Kit などの TUNEL法です。
What is the excitation/emission maxima of the Alexa Fluor 488 dye?
Alexa Fluor 488 has fluorescence excitation and emission maxima of 495/519 nm.
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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.
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I need to test cells for apoptosis after they have been formaldehyde-fixed and permeabilized. What dye or conjugate do you recommend? Will Annexin V conjugates work?
We do not recommend Annexin V for post-fix labeling, since fixation inactivates the function of the translocase; fixed samples would show mostly uniform labeling with Annexin V. The only options you have for apoptosis assays after fixation are to use an anti-caspase antibody or perform a TUNEL assay, such as with the Click-iT TUNEL Imaging kits.
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Can I use Click-iT TUNEL Alexa Fluor Imaging Assays for Microscopy & HCS (Cat. No. C10246) for flow cytometry?
We have not validated the use of Click‐iT TUNEL assay for flow cytometry. Theoretically, any Click‐iT TUNEL assay for imaging can be adapted to be used with flow cytometry. In general, follow the protocol as provided but spin down the suspension cells after every step. Start with about 10∧6 cells at about 10∧7 cell/mL. Please note that flow cytometry is more sensitive than fluorescence imaging, so you should use between 1/5th to 1/10th of the azide dye detection reagent in the click reaction. All other concentrations of the click reaction reagents should stay the same. We recommend using the Click‐iT Plus TUNEL assays (C10617, C10618, C10619), as the detection reagent is provided in a separate vial, enabling you to modify the concentration used.
The Click‐iT Plus TUNEL assay protocol can be found on the following link.
Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.