Apoptosis assay cell type - HeLa cells

A gross majority of classical apoptotic attributes can be quantitatively examined by flow cytometry, the preferred platform for rapid assessment of multiple cellular attributes at a single-cell level. However, sample preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

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

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream 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.

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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

Guava Nexin Annexin V Assay

Millipore

Upstream tips	Protocol tips	Downstream tips
	Cells were seeded in 6-well tissue culture plates the night before siRNA transfections. At 24 h post-siRNA addition, the cells were treated with nutlin-3b or 3a for additional 24–96 h. Culture media were collected and combined with the trypsinized-attached cells. Cell pellets were collected by centrifugation at 2000 r.p.m. for 10 min at 4 °C. The percentage of apoptotic cells was determined by Annexin V/7-AAD staining using the Guava Nexin kit and the Guava personal cell analyzer (Guava Technologies, Hayward, CA, USA), and calculated as the total fraction Annexin V-positive cells as described (Thompson et al., 2004).

Protocol tips
Cells were seeded in 6-well tissue culture plates the night before siRNA transfections. At 24 h post-siRNA addition, the cells were treated with nutlin-3b or 3a for additional 24–96 h. Culture media were collected and combined with the trypsinized-attached cells. Cell pellets were collected by centrifugation at 2000 r.p.m. for 10 min at 4 °C. The percentage of apoptotic cells was determined by Annexin V/7-AAD staining using the Guava Nexin kit and the Guava personal cell analyzer (Guava Technologies, Hayward, CA, USA), and calculated as the total fraction Annexin V-positive cells as described (Thompson et al., 2004).

Protocol tips

Cells were seeded in 6-well tissue culture plates the night before siRNA transfections. At 24 h post-siRNA addition, the cells were treated with nutlin-3b or 3a for additional 24–96 h. Culture media were collected and combined with the trypsinized-attached cells. Cell pellets were collected by centrifugation at 2000 r.p.m. for 10 min at 4 °C. The percentage of apoptotic cells was determined by Annexin V/7-AAD staining using the Guava Nexin kit and the Guava personal cell analyzer (Guava Technologies, Hayward, CA, USA), and calculated as the total fraction Annexin V-positive cells as described (Thompson et al., 2004).

Manufacturer protocol Publication protocol 1 Relevant paper

FITC Annexin V Apoptosis Detection Kit I

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
- 1×10^5 cells/ml cells were seeded for an assay. - BAK cells were added to HeLa cells after 6h and incubated for 20h.	- Treated with FITC Annexin V and PI and incubated for 15 min at RT (25°C) in the dark

Upstream tips
- 1×10^5 cells/ml cells were seeded for an assay. - BAK cells were added to HeLa cells after 6h and incubated for 20h.
Protocol tips
- Treated with FITC Annexin V and PI and incubated for 15 min at RT (25°C) in the dark

Manufacturer protocol Publication protocol 1 Relevant paper

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