1. Harvest cells - refer to specific procedures for corresponding cell lines or primary cell isolations.
2. Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.
3. Resuspend cells in 2 mL 1 x phosphate buffered saline (PBS) ^-/- (no calcium, no magnesium).
4. Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.
5. Resuspend cells in 1 mL 1 x Annexin V binding buffer.
6. Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.
7. Resuspend cells in 100 μL 1 x Annexin V binding buffer.

1. Add Annexin V according to the manufacturer's recommendations [e.g. 5 μL Annexin V Alexa Fluor 488 (Molecular Probes, A13201)].
2. Incubate tubes in the dark for 15 minutes at room temperature.
3. Add 100 μL of 1 x Annexin V binding buffer to each reaction tube. There should be approximately 200 μL in each tube.
4. Add 4 μL of PI (Sigma, Cat# P-4864-10ML) that has been diluted 1:10 in 1 x Annexin V binding buffer (i.e. 1 μL PI with 9 μL 1 x Annexin V binding buffer). This will yield a final PI concentration of 2 μg/mL in each sample.
5. Incubate tubes in the dark for 15 minutes at room temperature.
6. Add 500 μL 1 x Annexin V binding buffer to wash the cells.
7. Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.
8. Resuspend cells in 500 μL 1 x Annexin V binding buffer and 500 μL 2% formaldehyde to create a 1% formaldehyde (fixative) solution. Mix tubes by gentle flicking.
9. Fix samples on ice for 10 minutes. Alternatively, samples can be stored overnight at 4°C in the dark. If the latter method is chosen, make sure to be consistent across all tubes labeled with Annexin V/PI (including compensation controls).
10. Add 1 mL 1 x PBS^-/- to each sample and mix gently by flicking.
11. Centrifuge tubes at 425 x g for eight minutes and decant the supernatant.
12. Repeat steps 2.10 and 2.11.
13. Resuspend pellet by flicking the tube.
14. Add 16 μL of 1:100 diluted RNase A (Sigma, R4642) to give a final concentration of 50 μg/mL. Incubate for 15 min at 37°C.
15. Add 1 mL 1 x PBS^-/- and mix gently by flicking.
16. Centrifuge tubes at 425 x g for eight minutes.
17. Samples are now ready to be analyzed. Alternatively, samples can be used for subsequent staining steps if Annexin/PI staining is being performed in parallel with other procedures.

Examples of cell types and cell lines that have varying degrees of false-positive PI staining are shown in Figure 1. To account for the false-positive events, cells were fixed and then treated with RNase A. This results in a significant decrease in the number of false-positive events, compared to cells that didn't undergo RNase treatment (Figure 2B). Figure 2C shows representative images of cells that underwent RNase treatment, compared to cells that had no RNase treatment. Figure 3 shows how the modified Annexin V/PI protocol, with fixation and RNase treatment steps, improves upon conventional protocols by limiting the number of false-positive staining events.

Figure 1. Conventional propidium iodide staining protocols lead to a significant number of false positive events. We have previously evaluated extent of false positive staining across primary cells across a broad range of animal models as well as in a variety of cell lines ¹⁰. Representative images show extent of false positive staining in three unique cell populations (one commonly used cell line - RAW macrophages and two primary cells - murine bone marrow macrophages (BMM) and goldfish primary kidney macrophages (PKM)). True positive events display the expected co-localization between PI and DRAQ5 within the nuclear compartment (yellow areas depict colocalization between PI and DRAQ5). DRAQ5 is highly membrane permeable dye that accurately stains nuclear compartment in both live and dead cells ^10,11,12. For easier visual determination of colocalization DRAQ5 stain was given a green pseudocolour.

Figure 2. Improving the accuracy of PI nuclear staining. (A) We assessed accuracy of PI nuclear staining based on degree of similarity of a number of well-characterized nuclear stains (BrdU, 7-AAD and DAPI) to DRAQ5. BrdU is a synthetic nucleoside that is incorporated into the DNA of proliferating cells, and as such will only stain within the nuclear compartment. 7-AAD has a high affinity for DNA and, similar to PI, cannot cross an intact plasma membrane. DAPI also has a strong affinity for DNA and is the most commonly used nuclear stain in fluorescent microscopy. The degree of similarity was >99% between BrdU, 7-AAD, DAPI and DRAQ5, highlighting their ability to accurately stain the cell nucleus in RAW 264.7 macrophages. In contrast, cytoplasmic PI staining based on conventional protocols leads to a marked decrease in percent similarity of staining relative to DRAQ5. (B) Similar results are observed in two primary cells tested: murine bone marrow macrophages (BMM) and goldfish primary kidney macrophages (PKM). Incorporation of 50 μg/mL RNase A at specific stage within the staining procedure removes non-nuclear PI staining and significantly increases degree of similarity relative to DRAQ5 staining. (C) Representative images of primary kidney macrophages show the degree of similarity for cells with and without RNase treatment. For easier visual determination of differences between treatments, DRAQ5 was given a green. Yellow areas depict colocalization between BrdU and DRAQ5, and PI and DRAQ5.

Figure 3. Modified Annexin V/PI significantly reduces false apoptosis/necrotic events. Primary goldfish kidney macrophages (PKM) were stained with conventional and modified Annexin V/PI protocols. Scatterplots depict the Annexin V/PI stain in goldfish PKM. The scatterplot on the left shows conventional Annexin V/PI staining (no RNase) of PKM cells. The scatterplot on the right shows PKM cells stained with the modified Annexin V/PI protocol (with RNase). The addition of RNase results in a marked reduction of PI staining due to the removal of false positive stains. PKM cells were then analyzed based on distinct populations within the cultures- early progenitors (EP), monocytes (Mono) and mature macrophages (Mat mac). The reduction in the number of positive PI events, due to removal of false positive events is more pronounced in large mature macrophage cells than in smaller early progenitor cells. Conclusions based on conventional protocols would have erroneously suggested a positive correlation in cellular death for more mature macrophage subsets.