Annexin v binding buffer recipe

In the Laboratory for Cellular and Molecular Immunology, headed by Dror Mevorach, MD, in the Hadassah-Hebrew College Clinic, Israel. mevorachd@hadassah.org.il

Contributed by Uriel Trahtemberg

• This protocol is suggested for using annexin V staining without biasing the samples.
• We've printed the outcomes that brought for this protocol within the journal “Apoptosis” (Trahtemberg et al. 2007).
• This protocol assumes concurrent staining with propidium iodide (PI), but PI could be prevented or exchanged for other dyes or stains when needed.
• We've used this protocol with excellent recent results for a large number of stains in tens as well as countless experiments. We study human and murine immune cells, mostly primary.

Protocol 1: Annexin V staining preliminary formulations

1. Prepare binding buffer (BB): 140 mM NaCl, 4 mM KCl, .75 mM MgCl 2 and 10 mM HEPES in DDW. Make use of this BB for those reactions. Please be aware that vendor provided binding buffers could be dangerous towards the cells when calcium is added, particularly if they contain phosphate. A number of them don't contain Mg or K, that are essential ions. To learn more, please make reference to the content.
2. Prepare an 85 mM CaCl 2 in DDW stock solution. Make use of this means to fix add calcium ten minutes before purchase of annexin V. For instance, to achieve your final power of 1 mM calcium, use 1.2 µL of calcium stock per 100 µL of cells to achieve 1.5 mM calcium, add 1.75 μL of calcium stock per 100 μL of cells.
3. Using flow cytometry, titrate annexin V from the usual quantity of cells you’ll be utilising inside your experiments, in the appropriate volume. Typical values are .2-.3 × 10 6 cells in 200 μL of BB. Use 2.5 mM calcium, and see the very best power of annexin V per 100 μL. Add annexin V only ten minutes before actual acquisition.
4. To ensure the calcium concentration required for the particular cell and model getting used, prepare 8 tubes of cells as in the last step. Stain all of them with annexin V as determined in the last step, using , .5, 1, 1.25, 1.5, 1.75, 2, and a pair of.5 mM calcium. Add annexin V and calcium only ten minutes before acquisition.

Protocol 2: Annexin V staining experimental protocol

1. Only use binding buffer (BB).
2. After using the cells in the culture have them on ice whatsoever occasions.
3. Make a stock solution of annexin V and PI in BB, to ensure that every sample will get 10 μL in the stock. Use annexin V as determined in protocol 1. We normally use .75 1.25 μg/mL PI, however, you should titrate your personal cells/model.
4. Make a 10 μL solution with annexin V for any single stain, and the other 10 μL solution with PI for any single stain. These will be utilized for single-stain controls for compensation determination, as needed.
5. Filter cells into acquisition tubes, place the tubes on ice, and proceed immediately towards the flow cytometer.
6. 10 mins before actual acquisition, add 10 μL in the annexin V and PI stock, and add calcium when needed in the 85 mM stock.
7. If several samples should be acquired, add some reagents having a time delay to prevent longer incubation occasions.
8. Unstained samples ought to be acquired utilizing the same calcium concentration as annexin V stained samples.
9. For further verification (as needed), stain one sample with PI but without calcium, to check light scatter and PI positive percentages using the cells that did receive calcium.