Bromodeoxyuridine Pulse Labelling Assay: A Technique to Measure Cell Proliferation

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. BrdU Stock Solution
   1. Prepare 32.5 mM BrdU (10 mg/ml) in Dulbecco's Phosphate Buffered Saline (DPBS).
3. BrdU Complete RPMI
   1. Add 6.2 µl of BrdU stock solution to 10 ml of Complete RPMI.
4. DNase Solution
   1. Prepare 1 mg DNase/ml in DPBS.
5. Staining Buffer
   1. Prepare 3% heat-inactivated FCS and 0.09% sodium azide in DPBS.
6. Refer to Materials List for definitions of Fixation Buffer, Permeabilization Buffer and Wash Buffer.

2. Cells

NOTE: Cells were not cultured for greater than 6 months. This method is directly adaptable to any non-adherent cell line with adjustments to cell density and culture media. Use cells that are growing exponentially at the initiation of the experiment.

1. Maintain NALM6 cells in T-75 culture flasks in Complete RPMI. Perform all steps under sterile conditions using a Class II Biosafety Cabinet.
2. Maintain NALM6 cells between 1-2 x 10⁶ cells per ml by splitting the culture thrice weekly.
3. Incubate at 37 °C in 5% CO₂ in air.

3. Pulse Labeling of Cells with BrdU

CAUTION: Handle BrdU with care as it is a potential mutagen and teratogen.

1. Centrifuge cells at 150 x g for 5 min.
   NOTE: Transferring cells into fresh media improves the reproducibility of the results.
2. Perform a cell count and resuspend cells in Complete RPMI at 2 x 10⁶ cells/ml.
3. Dilute cells 1 in 2 with BrdU Complete RPMI producing a final cell concentration of 1 x 10⁶ cells/ml.
4. Incubate at 37 °C with 5% CO₂ for 45 min, then dilute cells 1 in 10 with complete RPMI. Centrifuge cells at 150 x g for 5 min and carefully discard all of the supernatant.
5. Resuspend cells in a small volume (~100 µl) of complete RPMI, perform a cell count and adjust to 1 x 10⁶ cells/ml.
6. Pipette 1 ml of cells into the wells of a 48 well plate. Pipette 1 ml of DPBS into any unoccupied wells to obtain more reproducible results.
7. Incubate at 37 °C in 5% CO₂ in air for desired timepoints, here 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hr.
   NOTE: The length of time will depend on what the experimental design aims to measure.
8. Transfer all the cells into FACS tubes using a pipette. Rinse the well sequentially with 1 ml volumes of PBS to a final total volume of 5 ml.
9. Centrifuge at 150 x g for 5 min and carefully remove all the supernatant. Cells are ready for staining, perform this (section 4) immediately.

4. 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.