BrdU Immunofluorescence Staining: A Technique to Identify Cells in Different Phases of Cell Cycle

1. Solutions and Reagents

1. Complete RPMI
   1. Add 56 ml fetal calf serum (FCS) and 5.5 ml of 200 mM L-glutamine to a 500 ml bottle of RPMI-1640 medium.
2. DNase Solution
   1. Prepare 1 mg DNase/ml in DPBS.
3. Staining Buffer
   1. Prepare 3% heat-inactivated FCS and 0.09% sodium azide in DPBS.
4. Refer to Materials List for definitions of Fixation Buffer, Permeabilization Buffer and Wash Buffer.

2. Cell Staining

NOTE: If surface staining of cells is required perform it prior to fixation, ensuring that the cells are kept at 4 °C throughout.

1. Resuspend cells in 100 µl of staining buffer (for optional surface staining, add the recommended volume of antibody to surface antigens and incubate for 30 min at 4 °C).
2. Add 1 ml of staining buffer, centrifuge for 5 min at 150 x g and discard the supernatant.
   NOTE: Specific antibody, concentration, incubation time etc. will vary depending on specific experimental goals.
3. Fixation and Permeabilization
   1. Resuspend cells in 100 µl of fixation buffer and incubate for 15 min at room temperature.
   2. Add 1 ml of wash buffer, centrifuge for 5 min at 150 x g and discard the supernatant.
   3. Resuspend cells in 100 µl of permeabilization buffer and incubate the cells for 10 min on ice.
   4. Add 1 ml of wash buffer, centrifuge for 5 min at 150 x g, and discard the supernatant.
   5. Resuspend cells in 100 µl of fixation buffer per tube and incubate for 5 min at room temperature.
   6. Add 1 ml of wash buffer, centrifuge for 5 min at 150 x g, and discard the supernatant.
      NOTE: The protocol can be paused here if required. The fixed cells are stable for several days at 4 °C if resuspended in staining buffer. Remove the staining buffer following centrifugation before proceeding.
4. DNase Treatment
   1. Resuspend cells in 100 µl of DNase solution (30 µg of DNase/10⁶ cells) and incubate cells for 1 hr at 37 °C.
   2. Add 1 ml of wash buffer, centrifuge at 150 x g for 5 min and discard supernatant.
5. Antibody Staining
   NOTE: Staining for intracellular markers other than BrdU can be performed simultaneously with the BrdU staining.
   1. IMPORTANT: Prepare compensation controls consisting of unstained cells and cells labeled with each single fluorochrome. Ideally, use the same antibodies for compensation controls as those used in the experimental tubes. However, if this is not feasible, substitute antibodies to highly expressed antigens conjugated to the same fluorochrome.
   2. Resuspend the cells in 50 µl of wash buffer and add 1 µl/10⁶ cells of BrdU antibody.
      NOTE: Directly conjugated antibodies to other specific intracellular antigens can also be added.  
      NOTE: Antibodies to histone H3 phosphorylated on Ser10 can be used to discriminate between cells in G2 and M, histone H3 is phosphorylated on Ser10 during mitosis. Antibodies to cdc2 phosphorylated on Tyr15 can be used to detect cells that have committed to mitosis.
   3. Incubate the cells for 20 min at room temperature.
   4. Add 1 ml of wash buffer, centrifuge cells at 150 x g for 5 min and discard supernatant.

6. Stain DNA for Cell Cycle Analysis
   1. Loosen pellet and add 20 µl of the 7-AAD solution (0.25 µg).
      NOTE: It is critical to use a constant amount of 7-AAD/cell.
   2. Resuspend the cells in 1 ml of Staining buffer.