Mammalian Cell Cycle Techniques

PBLs Isolation And Stimulation With PHA

1. Add 1 ml Heparin to 1 unit of blood (50 ml), mix gently.

2. Transfer blood to a tissue culture flask and dilute 4 fold with RPMI to a final volume of 200 ml.

3. Put a 12 ml aliquot of Ficol in several 50 ml Falcon tubes. Carefully layer 30 ml of the blood/RPMI mix on the top of the Ficol.

4. Spin at 2200 rpm in a table top centrifuge for 40 minutes at room temperature.

5. Remove serum (on the top) with a Pasteur pipette hooked up to vacuum.

6. Collect the yellowish cell layer, about 3 to 7 mls per tube (avoid red cells).

7. Resuspend every 10 ml of cells in 40 ml RPMI + 10% FCS (heat inactivated), spin at 1000 rpm for 10 minutes to pellet.

8. Pool cells together, wash cells with 50 ml of RPMI + 10% FCS once more.

9. Resuspend cell pellets in 50 ml of RPMI + 10% FCS, transfer to two 150 cm flasks, incubate for 1 hour to deplete monocytes which attach to the bottom.

10. Pool unattached cells and count cell number, add RPMI +10% FCS to 2 x 106 cells/ml. Take 50 ml out as a no PHA control.

11. Add PHA to 1mg/ml final, split cells into several flasks and incubate for desired time.

12. Harvest cells at different time for isolation of RNA or protein. Remember to use 2-4 x 106 cells for FACS analysis.

Cell synchronization using a double thymidine block

1. Cells are grown to a subconfluent density (mid-log phase) in serum-rich medium.

2. Thymidine is added to 2 mM final and incubated for 16 hours for HEp-2, 19 h for HeLa.

3. Cells are washed three times with PBS on plates and refed with fresh serum-rich medium.

4. Incubate for 10 hours before adding 2 mM thymidine.

5. Incubate for 14 hours (17 for HeLa) and wash as described in step 3, refeed, and begin time points.

Note: Conditions for synchronization of different cell lines may vary (e.g. time of incubation).

Cell Cycle Analysis (anti-BrdU-FITC)

1. Seed 10- or 15-cm plates with cells in media supplemented with dialyzed serum (regular FBS fine) 24 h before treatment. (Seed enough cells so that you end up with approximately 2.0×106 cells per plate at harvest time, but prevent contact inhibition.)

2. Treat cells as desired. Minimize exposure to light from this point on.

3. At the desired time point(s), pulse-label the cells with 10 micromolar BrdU for 30 minutes from a 100X stock solution (1 mM or 0.307 mg per ml PBS). Alternatively, continuously-label cells with 65 micromolar BrdU from a 100X stock solution (6.5 mM or 2 mg per ml PBS).

4. Aspirate medium and wash cells once with PBS-. Trypsinize cells, minimizing the time in trypsin. Transfer cells to a 15-ml centrifuge tube and spin at ~1,000 rpm for 3 minutes. Aspirate supernatant and re-suspend cells in 200 microliters PBS-.

5. Add 5 ml fresh ice cold 70% EtOH dropwise while vortexing the cells at setting #4. Allow cells to fix overnight. (Fixed cells should not be stored more than a week.)

6. Start boiling water bath. Centrifuge cells and aspirate supernatant. Resuspend cells in 1 ml cold 0.1M HCl/0.5% Triton X-100. Incubate for 10 minutes on ice. Add 5 ml dH20. Centrifuge cells and aspirate supernatant.

7. Resuspend cells in 2 ml dH2O. Place in boiling water bath for 10 minutes, then quickly transfer to ice for 5 minutes to cool.

8. Add 5 ml PBS-/0.5% Triton X-100. Centrifuge cells, aspirate supernatant, and resuspend cells.

9. Make 5 microgram/ml anti-BrdU-FITC solution (100 microliters per sample): (1:20 dilution of 100 microgram/ml stock diluted with 0.1% BSA in PBS-)

10. Resuspend cells in 100 microliters antibody solution. Incubate 30 mintues at room temperature in the dark, mixing every 5 to 10 minutes.

11. Cut filter squares and syringes. Make PI stain (5 micrograms/ml PI + 200 micrograms/ml RNase): (12.5 microliters 2 mg/ml PI + 100 microliters 10 mg/ml RNase + 4.9 ml PBS-)

12. Add 5 ml PBS- to cells. Centrifuge and aspirate supernatant.

13. Resuspend pellet in 250-500 microliters PI stain, depending on number of cells (optimally, each sample will be at a final concentration of 1×106 cells per ml).

14. Filter final solution through mesh filter on 1 cc syringes into Falcon 2052 tubes. Store at 4C for at least 10 minutes, but not more than a couple hours


15. FACScan.