Protocol of Cell Cycle Staining Flow Cytometry

Provided here are protocols for staining using DNA binding dyes with and without antibody staining and a protocol for BrdU staining. It should be noted that these are guidelines only. The incubation times may need to be adjusted for different cell types and different antibodies. Please refer to our Troubleshooting of Cell Cycle Staining Flow Cytometry when you are in trouble. If necessary, please don’t hesitate to contact us for additional help.

Refer to the corresponding sections according to your experimental requirements.

Contents:

Protocol A: Protocol of immunofluorescence staining of cells in combination with propidium iodide staining of cells for cell cycle analysis

Protocol B: Protocol of propidium iodide staining of cells for cell cycle analysis

Protocol C: Protocol of BrdU staining of cells for cell cycle analysis

Protocol A: Protocol of immunofluorescence staining of cells in combination with propidium iodide staining of cells for cell cycle analysis

Reagents

• PBS
• 70% ethanol
• 2% (w/v) paraformaldehyde in PBS
• 0.1% saponin (w/v)
• Propidium iodide (PI)
• Nucleic acid staining solution (1x PBS, 100 ug/mL RNAse A)

Methods

1. Prepare cells appropriately. Please refer to Cell Preparation for Flow Cytometry.
2. Fix in 2 mL 2% paraformaldehyde for 30 min on ice.
3. Centrifuge at 500 x g for 5 min. Discard supernatant.
4. Resuspend in 5 mL pre-cold 70% ethanol. Add dropwise to cell pellet while vortexing. Fix for at least 30 min on ice.
(Specimens can be left at this stage for several weeks.)
5. Centrifuge at 500 x g for 5 min. Discard supernatant.
6. Wash twice with 0.1% saponin at 400 g at 4°C for 5 min. Discard the supernatant.
7. Resuspend in 100 mL of 0.1% saponin. Add the directly conjugated antibody at the vendor-recommended dilution and incubate for at least 30 min at 4°C. Avoid direct light.
8. Wash twice in PBS. Pellet cells at 400 g at 4°C for 5 min. Discard supernatant.
9. Resuspend in 500 uL nucleic acid staining solution and incubate for 30 min at RT. Avoid direct light.
10. Add appropriate PI (1-2 drops).
11. Analyze cells by flow cytometry.
12. Data analysis.

Notes

1. Protocol A only provides a general procedure for DNA staining for cell cycle analysis using PI when you need to stain for other intracellular antigens.
2. Appropriate controls should always be carried out. For flow cytometry, the following should be considered for inclusion.
• Isotype controls used to determine if the staining is specific.
• Unstained cells should always be included in the experimental set-up to monitor autofluorescence.
3. For all multi-color flow cytometry experiments, it is suggested to include compensation controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating boundaries.

Protocol B: Protocol of propidium iodide staining of cells for cell cycle analysis

Reagents

• PBS
• 70% ethanol
• Nucleic acid staining solution (1x PBS, 100 ug/mL RNAse A)
• Propidium iodide (PI)

Methods

1. Prepare cells appropriately. Please refer to Cell Preparation for Flow Cytometry.
2. Fix in 5 mL pre-cold 70% ethanol. Add dropwise to cell pellet while vortexing. Fix for at least 30 min on ice.
(Specimens can be left at this stage for several weeks.)
3. Centrifuge at 500 x g for 10 min. Discard supernatant.
4. Wash twice with 3 mL PBS at 400 g at 4°C for 5 min. Discard supernatant.
5. Resuspend cell pellet in 500 uL nucleic acid staining solution. Mix thoroughly.
6. Incubate for 30 min at RT.
7. Add appropriate PI (1-2 drops).
8. Analyze cells by flow cytometry.
9. Data analysis.

Notes

1. For Protocol B, unstained cells should always be included in the experimental set-up to monitor autofluorescence.

Protocol C: Protocol of BrdU staining of cells for cell cycle analysis

Reagents

• PBS
• PBS-BSA: PBS containing 1% BSA
• 0.05% (v/v) Tween 20 in PBS-BSA
• 0.1M Na₂B₄O₇, pH 8.5
• 2M HCl containing 0.5% Triton X-100
• Propidium iodide (PI)

Methods

1. Add BrdU to the cell suspension in culture medium to a final concentration of 10 μM. Incubate for at least 30 min at 37°C in a CO₂ incubator.
2. Wash cells twice with PBS-BSA at 500 x g for 10 min at RT. Discard supernatant.
3. Resuspend in 5 mL pre-cold 70% ethanol. Add dropwise to cell pellet while vortexing. Fix for at least 30 min on ice.
4. Centrifuge at 500 x g for 10 min. Discard supernatant.
5. Resuspend the pellet in 2 mL 2 M HCl containing 0.5% Triton X-100. Incubate for 30 min at RT
(This incubation step is preferably performed on a rocking platform.)
6. Centrifuge at 500 x g for 10 min. Discard supernatant. Resuspend in 3 mL 0.1 M Na₂B₄O₇, pH 8.5 for 2 min.
7. Centrifuge at 500 x g for 10 min. Discard supernatant. Resuspend in PBS-BSA containing 0.05% Tween 20.
8. Adjust cell concentration to 1x10⁷ cells/mL. Aliquot 100 μL of the cell suspension into required number of flow cytometry tubes.
9. Incubate with antibody at the recommended vendor dilution overnight at 4°C. Avoid direct light.
10. Resuspend in 2 mL PBS-BSA. Centrifuge at 500 x g for 10 min.
11. If a secondary antibody is required, then discard the supernatant, add 100 μL of PBS-BSA and incubate with the secondary antibody at the vendor recommended dilution for at least 30 min at 4°C.
12. Wash with 2 mL PBS-BSA. Centrifuge at 500 x g for 10 min.
13. Resuspend cells in 1 mL PBS.
14. Add appropriate PI (1-2 drops).
15. Analyze cells by flow cytometry.
16. Data analysis.

Notes

1. The acid treatment to unwind the DNA may affect surface immunophenotyping. Staining of cells with BrdU using DNAse I may be applicable if this is required.
2. Appropriate controls should always be carried out. For flow cytometry, the following should be considered for inclusion.
• A known positive sample
• Isotype controls used to determine if the staining is specific
• Unstained cells should always be included in the experimental set-up to monitor autofluorescence
3. For all multi-color flow cytometry experiments, it is suggested to include compensation controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating boundaries.