Publication protocol
Flow Cytometry Protocol:
Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of inducing agent.
Harvest the cells after the incubation period and wash in cold phosphate-buffered saline (PBS).
Prepare 1X annexin-binding buffer. For example, for ~10 assays, add 1 mL 5X annexin binding buffer (Component C) to 4 mL deionized water.
Prepare a 100 µg/mL working solution of PI by diluting 5 µL of the 1 mg/mL PI stock solution (Component B in 45 µL 1X annexin-binding buffer. Store the unused portion of this working solution for future experiments.
Re-centrifuge the washed cells (from step 2), discard the supernatant and resuspend the cells in 1X annexin-binding buffer. Determine the cell density and dilute in 1X annexin-binding buffer to ~1 × 106 cells/mL, preparing a sufficient volume to have 100 µL per assay.
Add 5 µL Alexa Fluor® 488 annexin V (Component A) and 1 µL 100 µg/mL PI working solution (prepared in step 4) to each 100 µL of cell suspension
Incubate the cells at room temperature for 15 minutes.
After the incubation period, add 400 µL 1X annexin-binding buffer, mix gently and keep the samples on ice.
As soon as possible, analyze the stained cells by flow cytometry, measuring the fluorescence emission at 530 nm (e.g., FL1) and >575 nm (e.g., FL3).
The population should separate into three groups: live cells show only a low level of fluorescence, apoptotic cells show green fluorescence, and dead cells show both red and green fluorescence. Confirm the flow cytometry results by viewing the cells under a fluorescence microscope, using filters appropriate for fluorescein (FITC) and tetramethyl rhodamine (TRITC) or Texas Red® dye.
Imaging Microscopy Protocol:
This protocol was developed using Jurkat cells treated with calprotectin to induce apoptosis and may be adapted for adherent cell lines.
Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of the inducing agent.
After the incubation period, wash the cells in cold PBS.
Prepare 1X annexin-binding buffer. For example, to make 1 mL 1X buffer, add 200 µL 5X annexin-binding buffer (Component C) to 800 µL deionized water.
Prepare a 100 µg/mL working solution of PI by diluting 5 µL 1 mg/mL PI stock solution (Component B) in 45 µL 1X annexin-binding buffer. Store the unused portion of this working solution saved for future experiments.
Re-centrifuge the washed cells (from step 2), discard the supernatant, and resuspend the cells in 1X annexin-binding buffer. Determine the cell density and dilute in the annexin-binding buffer to ~1 × 106 cells/mL, preparing a sufficient volume for deposition on a slide.
Add 5–25 µL of the annexin V conjugate (Component A) and 1–2 µL of the 100 µg/mL PI working solution (prepared in step 4) to each 100 µL of cell suspension. Higher concentrations of the annexin V conjugate tend to produce better results; the optimal staining concentration needs to be determined empirically.
Incubate the cells at room temperature for 15 minutes.
Wash the cells with 1X Annexin-Binding buffer.
Deposit the cells onto slides, mount them using the desired method and observe the fluorescence using appropriate filters.
The cells should be separated into three groups: live, apoptotic, and dead. Live cells show only weak annexin V staining of the cellular membrane, while apoptotic cells show a significantly higher degree of surface labeling. Dead cells show both membranes staining by annexin V and strong nuclear staining from the propidium iodide.
Full paper
Login or
join for free to view the full paper.