Kit de ensayo de citometría de flujo Click-iT™ Plus EdU Alexa Fluor™ 350

El kit de ensayo de citometría de flujo Click-iT Plus EdU Alexa Fluor 350 proporciona un ensayo simplificado y másMás información

Número de catálogo	Cantidad
C10645	50 ensayos

Número de catálogo C10645

Precio (CLP)

Cantidad:

50 ensayos

El kit de ensayo de citometría de flujo Click-iT Plus EdU Alexa Fluor 350 proporciona un ensayo simplificado y más sólido para analizar la replicación de ADN en las células proliferantes en comparación con los métodos de BrdU tradicionales. El ADN recién sintetizado se analiza mediante el láser de UV del citómetro de flujo. La formulación Click-iT Plus es compatible con fluoróforos estándar, incluido los tándems R-PE y R-PE, así como con proteínas fluorescentes.

• Multiplexable: compatible con R-PE (y tándems) y proteínas fluorescentes
• Precisa: resultados superiores en comparación con ensayos BrdU
• Rápida: resultados en tan solo 60 minutos

Consultar la guía de selección para todos los ensayos de EdU Click-iT y EdU Click-iT Plus para citometría de flujo.

Multiplexable
Los ensayos multiplexables Click-iT Plus Alexa Fluor 350 EdU se pueden utilizar junto con tándems R-PE y R-PE, así como proteínas fluorescentes como la GFP y la mCherry. El reactivo de etiquetado Alexa Fluor 350 se excita fácilmente a 350 nm con un láser UV y emite a 438 nm.

Un método avanzado que ofrece resultados superiores a BrdU
El método más preciso de análisis de proliferación es la medición directa de la síntesis del ADN. Originalmente, este procedimiento se realizaba mediante la incorporación de nucleósidos radiactivos. Este método fue reemplazado por la detección basada en anticuerpos del análogo nucleósido bromodesoxiuridina (BrdU). El ensayo de citometría de flujo Click-iT Plus EdU es una novedosa alternativa al ensayo de BrdU. EdU (5-etinil-2´-desoxiuridina) es un análogo de la timidina que se incorpora al ADN durante la síntesis de ADN activa. La detección se basa en la química de clic, que es una reacción covalente catalizada con cobre entre una azida y un alquino. En esta aplicación, el alquino se encuentra en la fracción de etinil de EdU, mientras que la azida se acopla al tinte Alexa Fluor. Se utilizan métodos de citometría de flujo estándar para determinar el porcentaje de células de fase S en la población.

Unas condiciones moderadas permiten el uso con tintes de ciclo celular y anticuerpos
El tamaño reducido de la azida de la tinción permite la detección eficaz de los EdU incorporados utilizando condiciones moderadas, mientras que la fijación basada en aldehído estándar y la permeabilización de detergente son suficientes para que el reactivo de detección Click-iT Plus consiga acceso al ADN. Esto ocurre de manera diferente en los ensayos de BrdU donde se requiere la desnaturalización del ADN (mediante HCl, calor o digestión con ADNasa) para exponer la BrdU de forma que pueda detectarla un anticuerpo anti-BrdU. El procesamiento de las muestras para el ensayo de BrdU puede provocar una alteración de la señal de la distribución del ciclo celular, además de la destrucción de los sitios de reconocimiento de los antígenos cuando se utilice el método HCl. Por el contrario, el sencillo ensayo Click-iT Plus EdU es compatible con tintes de ciclo celular. El ensayo Click-iT Plus EdU también se puede multiplexar con anticuerpos contra la superficie y marcadores intracelulares, así como conjugados etiquetados con fluoróforos estándar incluidos tándems R-PE, R-PE y proteínas fluorescentes (GFP y mCherry).

Protocolo rápido y sencillo
El protocolo Click-iT Plus EdU se basa en los pasos de fijación mediante aldehído y permeabilización de detergente para el etiquetado de anticuerpos inmunohistoquímicos. Sin embargo, EdU es compatible con otros agentes de fijación/permeabilización incluidos el metanol y la saponina. En tan solo cinco pasos podrá empezar a analizar sus datos de proliferación celular:

1. Trate las células con EdU.
2. Fije y permeabilice las células.
3. Detecte células de fase S con el cóctel de detección Click-iT Plus durante 30 minutos.
4. Lave una vez.
5. Analice.

Los resultados se pueden comprobar en tan solo 60 minutos en algunas circunstancias, pero recomendamos 90 minutos para todas las aplicaciones.

Para uso exclusivo en investigación. No apto para uso en procedimientos diagnósticos.

Especificaciones

FormatoKit

Cantidad50 ensayos

Condiciones de envíoTemperatura ambiente

Emission350⁄438

Para utilizar con (equipo)Citómetro de flujo

Línea de productosAlexa Fluor, Click-iT

Tipo de productoReactivo

Unit SizeEach

Contenido y almacenamiento

Contiene EdU (5-etinil-2'-desoxiuridina), azida picolil AlexaFluor 350, dimetilsulfóxido anhidro (DMSO), fijador Click-iT, tampón de permeabilidad y lavado Click-iT a base de saponina, protector de cobre y aditivo de tampón Click-iT EdU.

Almacenar entre 2 °C y 8 °C

Desecar y proteger de la luz.

Preguntas frecuentes

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I am observing no signal or very low specific signal for my click-labeled samples. What can I do to improve the signal?

The click reaction is only effective when copper is in the appropriate valency. Azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest.
Do not use additive buffer that has turned yellow; it must be colorless to be active.
Cells need to be adequately fixed and permeabilized for the TdT enzyme and click reagents to have access to the nucleus. Tissue samples require digestion with proteinase K or other proteolytic enzymes for sufficient TdT access.
Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be o xidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction.
You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling.
Your cells may not be apoptotic. Prepare a DNase I-treated positive control to verify that the TdT enzymatic reaction and click labeling reaction are working correctly.

Find additional tips, troubleshooting help, and resources within our Labeling Chemistry Support Center.

I am observing high non-specific background when I image my Click-iT EdU TUNEL-labeled samples. What is causing this and what can I do to reduce the background?

The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no-click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You should also perform the complete click reaction on a no-TdT enzyme control sample to verify the specificity of the click reaction signal.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I notice that when I post-stain my cells with DAPI after performing the click reaction to detect EdU incorporation, my DAPI signal is lower compared to my no-click reaction control samples. What causes the reduction in DAPI signal?

The copper in the click reaction denatures DNA to a small extent (although not as much as is required for efficient BrdU detection), which can affect the binding affinity of DNA dyes including DAPI and Hoechst stain. This effect should only be apparent with the classic EdU kits and not the Click-iT Plus EdU kits, which use a lower copper concentration.

Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.

I am observing no signal or very low signal for my click-labeled samples. What can I do to improve the signal?

The click reaction is only effective when copper is in the appropriate valency. Except for the DIBO alkyne-azide reaction, azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest.
Do not use additive buffer that has turned yellow; it must be colorless to be active.
Cells need to be adequately fixed and permeabilized for the click reagents to have access to intracellular components that have incorporated the click substrate(s).
Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be oxidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction.
You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling.
Low signal can also be due to low incorporation of EdU, EU, or other click substrates. Other click substrates (e.g., AHA, HPG, palmitic acid, azide, etc.) incorporated into cellular components may have been lost if not adequately cross-linked in place or if the wrong fixative was used. For click substrates that are incorporated into the membrane or lipids, you should avoid the use of alcohol or acetone fixatives and permeabilizing agents.
The incorporated click substrate must be accessible at the time of the click reaction; labeling of incorporated amino acid analogs may be lower in native proteins relative to denatured proteins.
You may need to optimize the metabolic labeling conditions including analog incubation time or concentration. Cells that are healthy, not too high of a passage number and not too crowded may incorporate the analog better. You may create a positive control by including extra doses of the click substrate during multiple time points during an incubation time that spans or closely spans the doubling time of the cell type of interest.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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