Protocol: Propidium Iodide Staining of Cells for Cell Cycle Analysis in Combination with Direct Immunofluorescence Intracellular Staining

Reagents

• Phosphate Buffered Saline 10x (PBS) (BUF036A)
• 70% ethanol in distilled water
• Nucleic acid staining solution (PBS containing 100 μg/mL RNase A)
• ReadiDrop Propidium Iodide (1351101)
• 2% (w/v) paraformaldehyde in PBS
• 0.1% saponin (w/v), 1% (w/v) bovine serum albumin in PBS

Methods

1. Prepare cells appropriately. For information on preparing cells from tissues or tissue culture cell lines, or for isolating peripheral blood mononuclear cells from whole blood, refer to Protocols FC1, FC2, or FC3.
2. Aliquot 100 μL of the cell suspension into as many 5 mL tubes as required.
3. Fix in 2 mL 2% paraformaldehyde for 30 min on ice.
4. Centrifuge at 500 x g for 5 min and discard the supernatant.
5. Resuspend cells in 2–5 mL cold (4°C) 70% ethanol. Add dropwise to the cell pellet while vortexing. This ensures the fixation of all cells and minimizes clumping.
6. Incubate for at least 30 min on ice.
   Note: Fixed cells can be stored at 4°C for several weeks.
7. Centrifuge at 500 x g for 10 min and discard the supernatant.
8. Resuspend cells in 500 μL 0.1% saponin and centrifuge at 300–400 x g for 5 min at 4°C; discard the supernatant.
9. Repeat step 8.
10. Resuspend in 100 μL 0.1% saponin and add directly conjugated antibody at the dilution determined from a titration experiment or the vendor-recommended dilution. Mix well and incubate at 4°C for 30–60 min, avoiding direct light.
11. Add 3 mL 0.1% saponin, centrifuge at 300–400 x g for 5 min at 4°C, and discard the supernatant.
12. Add 3 mL PBS, centrifuge at 300–400 x g for 5 min at 4°C, and discard supernatant.
13. Resuspend cells in 500 μL nucleic acid staining solution and mix well.
    Note: The presence of RNase A ensures that no RNA is present that would otherwise also stain with propidium iodide.
14. Incubate for 30 min at room temperature, avoiding direct light.
15. Add 1–2 drops of ReadiDrop Propidium Iodide and vortex briefly.
16. Acquire samples on a flow cytometer.

Notes

• When acquiring cells, select the appropriate laser and filter for propidium iodide and collect data in the linear scale
• Doublets should be gated out during analysis using the forward scatter (FSC) area versus height or width, depending on your instrument
• Alternative DNA binding dyes that are also suitable for cell cycle analysis include ReadiDrop 7-AAD (7-aminoactinomycin-D) (1351102) and PureBlu Hoechst 33342 Nuclear Staining Dye (1351304)
• Include appropriate controls, such as unstained cells, to monitor background staining
• All antibodies should be titrated prior to use to ensure the optimal working concentration is used
• If a directly conjugated antibody is not available commercially, an alternative is to conjugate a purified format of the antibody using a conjugation kit. These are available in a wide range of formats, such as Bio-Rad TrailBlazer™ Tag and StarBright™ Dye Label Kits, LYNX Rapid Conjugation Kits, and Readilink Kits