Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI)

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	Click-ITTM EdU-Alexa Fluor488 Kit (Life Technologies) was used to assess cell proliferation. The 5-ethynyl-2’-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA only during DNA synthesis, allowing for the quantitation of newly synthesized DNA (Salic and Mitchison 2008; Hamelik and Krishan 2009). The use of EdU is a less toxic alternative to the bromodeoxyuridine method, as it does not require the DNA denaturation and harsh permeabilization steps. To perform the assay, sorted cells were treated with 2.5 mM EdU-Alexa Fluor 488 (Life Technologies), directly added to the culture medium, and processed at different time intervals according to manufacturer’s instructions.

Protocol tips

Click-ITTM EdU-Alexa Fluor488 Kit (Life Technologies) was used to assess cell proliferation. The 5-ethynyl-2’-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA only during DNA synthesis, allowing for the quantitation of newly synthesized DNA (Salic and Mitchison 2008; Hamelik and Krishan 2009). The use of EdU is a less toxic alternative to the bromodeoxyuridine method, as it does not require the DNA denaturation and harsh permeabilization steps. To perform the assay, sorted cells were treated with 2.5 mM EdU-Alexa Fluor 488 (Life Technologies), directly added to the culture medium, and processed at different time intervals according to manufacturer’s instructions.

Publication protocol

Annexin V Assay and Caspase 3/7 Staining
The Annexin V-Alexa Fluor 647 (Life Technologies, Grand Island, NY) staining procedure was performed according to the manufacturer’s protocol. Briefly, the cells were resuspended with the Annexin V-binding buffer to a concentration of 1×106 cells/ml and stained with Annexin V-conjugate. Samples were incubated for 20 min at room temperature in the dark. After washing with the Annexin V-binding buffer, the cells were analyzed with a flow cytometer.

For caspase 3/7 staining CellEventTM Caspase 3/7 Green reagent (Life Technologies), a fluorogenic substrate for activated caspase-3 and -7 was used according to the manufacturer’s protocol. The cells were incubated with 5 μM of substrate for 30 min at room temperature and then analyzed with a flow cytometer.

Click-IT Proliferation Analysis
Click-ITTM EdU-Alexa Fluor488 Kit (Life Technologies) was used to assess cell proliferation. The 5-ethynyl-2’-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA only during DNA synthesis, allowing for the quantitation of newly synthesized DNA (Salic and Mitchison 2008; Hamelik and Krishan 2009). The use of EdU is a less toxic alternative to the bromodeoxyuridine method, as it does not require the DNA denaturation and harsh permeabilization steps. To perform the assay, sorted cells were treated with 2.5 mM EdU-Alexa Fluor 488 (Life Technologies), directly added to the culture medium, and processed at different time intervals according to manufacturer’s instructions.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI) below.

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