BrdU Labeling of HeLa Cells Followed by Immunostaining

Reagent

5′-bromo-2′-deoxyuridine (BrdU)

Fetal bovine serum (FBS)

Hydrochloric acid (HCl)

Immunofluorescence buffer (IF buffer) (prepare by adding 0.75 g glycine per 100 ml phosphate buffered saline (PBS))

Paraformaldehyde (PFA)

PBS Triton X-100

Rat Anti-BrdU Antibody clone RF06 (MCA6144)

(Optional) PureBlu DAPI Nuclear Staining Dye (1351303) and antibody to stain cytoplasmic proteins, for example, Rabbit Anti-GAPDH Antibody (AHP1628)

Method

1. Place sterilized glass cover slips into two wells of a 6-well plate. Seed HeLa cells at a density of 1 x 10⁴
   cells/well in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS into the wells containing cover slips (5 ml of media per well). Allow the cells to settle for 24-48 hr or until approximately 80 % confluent.
2. Prepare 5 ml of BrdU treated medium by diluting 100 mM BrdU stock solution 1:1,000 in DMEM culture medium containing 10% FBS.
3. Prepare 5 ml of DMSO treated medium by diluting DMSO 1:1000 in DMEM culture medium containing 10% FBS.
4. Remove the culture medium from the cells by pipette. Add the 5 ml of media containing BrdU to the well of cells to be labeled with BrdU and the 5 ml of media containing DMSO to the well of cells that are to act as your negative control. Incubate BrdU and DMSO treated HeLa cells for 3 hr at 37°C.
5. Wash the cells three times (3x) with 2 ml of 1x PBS/well.
6. Add 1 ml of PBS with 4% PFA/well. Incubate for 15 min at RT.
7. Wash the cells 3x with 2 ml of 1x PBS/well.
8. Add 1 ml of 1% Triton X-100/well. Incubate for 10 min at RT.
9. Wash the cells 3x with 2 ml of 1x PBS/well.
10. Add 1 ml 2 M HCl /well. Incubate for 30 min at RT.
11. Wash the cells 3x with 2 ml of 1x PBS/well.
12. Wash cells once with 2 ml of IF buffer/well.
13. Add 1 ml of IF buffer to each well of cells. Incubate for 30 min at RT. Remove excess liquid by pipetting.
14. Add 1 ml of PBS with 10 % FCS to each well of cells. Incubate for 30 min at RT. Remove liquid by pipetting (do not wash off).
15. Make up 2 ml of primary antibody mixture by adding 4 µg of Rat Anti-BrdU Antibody (MCA6144) and 4 µg of Rabbit Anti-GAPDH Antibody (AHP1628) to 2 ml of IF buffer.  Add 1ml of primary antibody mixture to the well containing BrdU treated cells and 1 ml of primary antibody mixture to the well containing DMSO treated cells. Incubate at 4℃ overnight without agitation.
16. Wash slides 3x with 1 ml of IF buffer/well.
17. Make up 2 ml of secondary antibody mixture by adding 10 µg of Goat Anti-Rat IgG (H/L): TRITC (305003) and 4 µg of Goat Anti-Rabbit IgG: Dylight 488 Antibody to 2 ml of IF buffer. Add 1 ml of secondary antibody mixture to the well containing BrdU treated cells and 1 ml of secondary antibody mixture to the well containing DMSO treated cells. Incubate for 1 hr at RT protected from light and without agitation.
18. Add 1 ml of 0.5 µg/ml of PureBlu DAPI (1351303) diluted in PBS to each well of cells and incubate at RT for 2 min.
19. Wash cells 3x with 1 ml of IF buffer/well.
20. Wash cells twice in 1 x PBS/well.
21. Remove the coverslips from the 6-well plate and remove excess liquid with tissue. Bond the cover slips to microscope slides with mounting medium. Store at 4℃ protected from light until ready to image.