BrdU Products and Resources for Flow Cytometry

Reagents

0.05% (v/v) Tween 20 in phosphate buffered saline (PBS)

0.1 M Na₂B₄O₇, pH 8.5

2 M HCl containing 0.5% Triton X-100

PBS

PBS containing 1% bovine serum albumin (PBS/BSA)

Propidium iodide

Methods

1. Add BrdU to the cell suspension in culture medium to a final concentration of 10 μM and 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, decant supernatant.
3. Resuspend in 2-5 ml cold (4^oC) 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, decant supernatant.
5. Resuspend the pellet in 2 ml of 2 M HCl containing 0.5% Triton X-100. Incubate for 30 min at RT (preferably on a rocking platform).
6. Centrifuge at 500 x g for 10 min, decant supernatant. Resuspend in 3 ml of 0.1 M Na₂B₄O₇, pH 8.5 for 2 min at RT.
7. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend in RT PBS/BSA + 0.05% Tween 20. Adjust cell concentration to 1 × 10⁷ cells/ml.
8. Aliquot 100 μl of the cell suspension into required number of FACS tubes.
9. Incubate with antibody at the recommended vendor dilution overnight at 4^oC avoiding direct light.
10. Resuspend in 2 ml of RT PBS/BSA. Centrifuge at 500 x g for 10 min at RT.
11. If a secondary antibody is required, then decant 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^oC.
12. Wash with 2 ml of PBS/BSA, centrifuge at 500 x g for 10 min.
13. Re-suspend cells in 1 ml of PBS. Add propidium iodide e.g. 1-2 drops of ReadiDrop™ Propidium Iodide (1351101).
14. Analyze by flow cytometry. The propidium iodide should be read on the appropriate channel in the linear scale. Doublets should be gated out using the Area vs Height or Width depending on your instrument.

Notes

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.

Appropriate controls should be carried out for flow cytometry, consider including the following:

• A known positive sample
• Isotype controls (to determine if the staining is specific)
• Unstained cells (should always be included to monitor autofluorescence)

For all multicolor flow cytometry experiments include compensation controls and fluorescence minus one (FMO) controls, which assist with identifying gating boundaries.

Reagents

0.05% (v/v) Tween 20 in phosphate buffered saline (PBS)

0.1 M Na₂B₄O₇, pH 8.5

2 M hydrochloric acid (HCl) containing 0.5% Triton X-100

DNA stains/cell viability dyes, such as propidium iodide

Mouse Anti-BrdU Antibody (clone Bu20a, MCA2483)

PBS

PBS containing 1% bovine serum albumin (PBS/BSA)

Methods

1. Label cells with BrdU. Add BrdU to the cell suspension in culture medium to a final concentration of 10 µM and incubate for 30 min in a CO₂ incubator at 37°C.
2. Wash cells 2 times (2x) with PBS/BSA, centrifuge cells at 500 x g for 10 min, decant supernatant, and resuspend in a minimum volume of PBS.
3. Add cells slowly into 5 ml 70% ethanol chilled to -20°C, mix continuously by vortexing.
4. Incubate on ice for 30 min.
5. Centrifuge at 500 x g for 10 min, and decant supernatant.
6. Add 2 ml of 2 M HCl containing 0.5% Triton X-100, and incubate the cells for 30 min at room temperature (RT) on a rocking platform set to 15 rpm. Denaturation of the DNA by HCl is critical for successful BrdU staining. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend in 1 ml of 0.1 M Na₂B₄O₇, pH 8.5.
7. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend the cells in PBS/BSA plus 0.05 % Tween 20 to a concentration of 1 x 10⁷ cells/ml.
8. Aliquot 100 µl of the cell suspension into the required number of FACS tubes.
9. Incubate cells with Mouse anti-BrdU Antibody (clone Bu20a) at the recommended dilution for 1 hr at RT. Alternatively, incubation can be performed overnight at 4°C.
10. To wash, add 2 ml of PBS/BSA, and centrifuge the cells at 500 x g for 5 min.
    
    (Optional) When using an unconjugated Bu20a antibody format perform incubation with a secondary antibody. For this purpose decant the supernatant and perform secondary antibody incubation for 45 min at RT (refer to the respective datasheet about the recommended dilution). Wash cells by repeating step 10.
     
11. Decant the supernatant and add 0.5 ml of sheath fluid.
12. For total DNA staining, add DNA stains such as, ReadiDrop™ Propidium Iodide (1351101), ReadiDrop 7-AAD (1351102), or PureBlu™ Hoechst 33342 (1351304), to each tube.
13. Analyze cells by flow cytometry following the manufacturer’s instructions. The DNA stain should be read on the appropriate channel set to the height and linear scale and not log scale.

Notes

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.

Appropriate controls should be carried out for flow cytometry, consider including the following:

• A known positive sample
• Isotype controls (to determine if the staining is specific)
• Unstained cells (should always be included to monitor autofluorescence)

For all multicolor flow cytometry experiments include compensation controls and fluorescence minus one (FMO) controls, which assist with identifying gating boundaries.

Reagents

0.05% (v/v) Tween 20 in phosphate buffered saline (PBS)

0.1 M Na₂B₄O₇, pH 8.5

2 M hydrochloric acid (HCl) containing 0.5% Triton X-100

DNA stains/cell viability dyes, such as propidium iodide

Rabbit Anti-BrdU Antibody (AHP2405)

PBS

PBS containing 1% bovine serum albumin (PBS/BSA)

Methods

1. Label cells with BrdU. Add BrdU to the cell suspension in culture medium to a final concentration of 10 µM and incubate for 30 min in a CO₂ incubator at 37°C.
2. Wash cells 2 times (2x) with PBS/BSA, centrifuge cells at 500 x g for 10 min, decant supernatant, and resuspend in a minimum volume of PBS.
3. Add cells slowly into 5 ml 70% ethanol chilled to -20°C, mix continuously by vortexing.
4. Incubate on ice for 30 min.
5. Centrifuge at 500 x g for 10 min, and decant supernatant.
6. Add 2 ml of 2 M HCl containing 0.5% Triton X-100, and incubate the cells for 30 min at room temperature (RT) on a rocking platform set to 15 rpm. Denaturation of the DNA by HCl is critical for successful BrdU staining. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend in 1 ml of 0.1 M Na₂B₄O₇, pH 8.5.
7. Centrifuge at 500 x g for 10 min, decant supernatant, and resuspend the cells in PBS/BSA plus 0.05 % Tween 20 to a concentration of 1 x 10⁷ cells/ml.
8. Aliquot 100 µl of the cell suspension into the required number of FACS tubes.
9. Incubate cells with Rabbit anti-BrdU Antibody at the recommended dilution for 1 hr at RT. Alternatively, incubation can be performed overnight at 4°C.
10. To wash, add 2 ml of PBS/BSA, and centrifuge the cells at 500 x g for 5 min.
11. Perform incubation with a secondary antibody. For this purpose decant the supernatant and perform secondary antibody incubation for 45 min at RT (refer to the respective datasheet about the recommended dilution). Wash cells by repeating step 10.
12. Decant the supernatant and add 0.5 ml of sheath fluid.
13. For total DNA staining, add DNA stains such as, ReadiDrop™ Propidium Iodide (1351101),ReadiDrop 7-AAD (1351102), or PureBlu™ Hoechst 33342 (1351304), to each tube. 
14. Analyze cells by flow cytometry following the manufacturer’s instructions. The DNA stain should be read on the appropriate channel set to the height and linear scale and not log scale.

Notes

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.

Appropriate controls should be carried out for flow cytometry, consider including the following:

• A known positive sample
• Isotype controls (to determine if the staining is specific)
• Unstained cells (should always be included to monitor autofluorescence)

For all multicolor flow cytometry experiments include compensation controls and fluorescence minus one (FMO) controls, which assist with identifying gating boundaries.

Reagents

Anti-BrdU Antibody

100 mM BrdU stock solution

FACS buffer

2M HCl

Leucoperm Reagent (BUF09B)

ReadiDrop Propidium Iodide (1351101)

Secondary Antibody

Methods

1. Seed Jurkat cells at a density of 2 x 10⁶ cells/well in RPMI-1640 medium containing 10% FBS into a 6-well plate.
2. Add BrdU stock solution to each well of the 6-well plate so that you have a final concentration of 100 µM BrdU (for instance for 3 ml of media, add 3 µl of the 100 mM stock solution of BrdU). Incubate for 1 hr at 37°C.
3. Collect the treated cells in a 15 ml falcon tube. Wash cells twice with 15 ml FACS buffer, centrifuging the cells at 1300 rpm for 5 min at room temperature (RT) between each wash, and decanting the supernatant.
4. Resuspend the cells in 100 μl Leucoperm Reagent A (BUF09B). Incubate for 15 min at RT.
5. Wash cells twice with 15 ml FACS buffer, centrifuging the cells at 1700 rpm for 5 min at RT between each wash and decant the supernatant.
6. Resuspend the cells in 100 μl Leucoperm Reagent B (BUF09B). Incubate for 5 min at RT.
7. Wash cells twice with 1 ml FACS buffer, centrifuging the cells at 1700 rpm for 5 min at RT between each wash and decant the supernatant.
8. Resuspend the pellet in 0.5 ml of 2 M HCl. Incubate for 30 min at RT (preferably on a rocking platform).
9. Wash cells twice with 1 ml FACS buffer, centrifuging the cells at 1700 rpm for 5 min at RT between each wash and decant the supernatant.
10. Resuspend the pellet in FACS buffer to a final density of 5 × 10⁶ cells/ml.
11. Add 100 μl of cell suspension to a 5 ml FACS tube. Centrifuge the cells, discard the supernatant, and add the anti-BrdU antibody of choice at the required dilution. Incubate for
    1 hr at RT or 4^oC overnight, avoiding direct light.
12. Wash cells with 1 ml FACS buffer, centrifuging the cells at 1500 rpm for 5 min at RT between each wash and decant the supernatant.
13. Incubate with a secondary antibody for 30 min at RT, avoiding direct light.
14. Wash cells twice with 1 ml FACS buffer, centrifuging the cells at 1500 rpm for 5 min at RT between each wash and decant the supernatant.
15. Prepare FACS buffer with ReadiDrop Propidium Iodide (1351101) by adding 1 drop of ReadiDrop Propidium Iodide per 500 μl of FACS buffer. 
16. Add 200 μl of FACS buffer with ReadiDrop Propidium Iodide to the cells. Analyze by flow cytometry.

Notes

Appropriate controls should be carried out for flow cytometry, consider including the following:

• A known positive sample
• Isotype controls (to determine if the staining is specific)
• Unstained cells (should always be included to monitor autofluorescence)

For all multicolor flow cytometry experiments, include compensation controls and fluorescence minus one (FMO) controls, which assist with identifying gating boundaries.