Protocol for Annexin V Staining

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From the Laboratory for Cellular and Molecular Immunology, headed by Dror Mevorach, MD, at the Hadassah-Hebrew University Medical Center, Israel. mevorachd@hadassah.org.il

Contributed by Uriel Trahtemberg

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• This protocol is proposed for the use of annexin V staining without biasing the samples.
• We have published the results that led to this protocol in the journal “Apoptosis” (Trahtemberg et al, 2007).
• This protocol assumes concurrent staining with propidium iodide (PI), but PI can be avoided or exchanged for other dyes or stains as needed.
• We have used this protocol with excellent results for thousands of stains in tens and even hundreds of experiments. We study human and murine immune cells, mostly primary.

Protocol 1: Annexin V staining preliminary preparations

1. Prepare binding buffer (BB): 140 mM NaCl, 4 mM KCl, 0.75 mM MgCl2 and 10 mM HEPES in DDW. Use this BB for all reactions. Please note that vendor provided binding buffers can be harmful to the cells when calcium is added, especially if they contain phosphate. Some of them do not contain Mg or K, which are essential ions. For more information, please refer to the article.
2. Prepare an 85 mM CaCl2 in DDW stock solution. Use this solution to add calcium 10 minutes before acquisition of annexin V. For example, to reach a final concentration of 1 mM calcium, use 1.2 µL of calcium stock per 100 µL of cells; to reach 1.5 mM calcium, add 1.75 μL of calcium stock per 100 μL of cells.
3. Using flow cytometry, titrate annexin V against the usual number of cells you’ll be using in your experiments, at the appropriate volume. Typical values are 0.2-0.3 × 106 cells in 200 μL of BB. Use 2.5 mM calcium, and determine the best concentration of annexin V per 100 μL. Add annexin V only 10 minutes before actual acquisition.
4. To verify the calcium concentration needed for the specific cell and model being used, prepare 8 tubes of cells as in the previous step. Stain them with annexin V as determined in the previous step, using 0, 0.5, 1, 1.25, 1.5, 1.75, 2, and 2.5 mM calcium. Add annexin V and calcium only 10 minutes before acquisition.

Protocol 2: Annexin V staining experimental protocol

1. Use only binding buffer (BB).
2. After taking the cells from the culture keep them on ice at all times.
3. Prepare a stock solution of annexin V and PI in BB, so that every sample will receive 10 μL from the stock. Use annexin V as determined in protocol 1. We normally use 0.75 – 1.25 μg/mL PI, but you should titrate your own cells/model.
4. Prepare a 10 μL solution with annexin V for a single stain, and another 10 μL solution with PI for a single stain. These will be used as single-stain controls for compensation determination, if needed.
5. Filter cells into acquisition tubes, put the tubes on ice, and proceed immediately to the flow cytometer.
6. Ten minutes before actual acquisition, add 10 μL from the annexin V and PI stock, and add calcium as needed from the 85 mM stock.
7. If several samples are to be acquired, add the reagents with a time delay to avoid longer incubation times.
8. Unstained samples should be acquired using the same calcium concentration as annexin V stained samples.
9. For additional verification (if needed), stain one sample with PI but without calcium, to compare light scatter and PI positive percentages with the cells that did receive calcium.

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