Mastering It in One Article: Synchronizing the Cell Growth Cycle

After culturing cells, it is observed that even within a single cell culture, the division time of individual cells varies. Some cells have completed division and growth, while others are still in the initial phase of division. However, in most experiments, maintaining synchronized cell growth cycles is essential to ensure more accurate experimental data. Therefore, artificial intervention becomes necessary.

1.Principle of Mitotic Shake-off:

During the M phase, cells become round and exhibit reduced adhesion. Gently shaking or tapping the culture flask can cause the detachment of M-phase cells.

Procedure: 1. Cells in the logarithmic growth phase (density 5-8×10^5/mL)

2.Add nocardazole 40-100 ng/mL

3.Cultivate for 10-14 h

4.Centrifuge to collect (300×g, 5 min)

5. Wash twice with cold PBS (4°C)

[Key step: Low temperature promotes microtubule depolymerization and enhances M-phase arrest]

6.Wash once with 37°C warm medium

7.Resuspend in fresh complete medium and incubate at 37°C

8.Sample every 15-30 min for flow cytometry detection of pHH3 (M-phase marker)

9.When M-phase cells exceed 80%, the cells are synchronized in the M phase.

2.Principle of G0 phase induction (serum starvation method): Deprivation of growth factors causes cells to exit the cycle and enter the G0 phase.

1.Collect cells in the logarithmic growth phase;

2.Cultivate in serum-free medium for 24 h. Note: Complete removal of cells may lead to death. Therefore, cells can be cultured in medium containing 1%-2% FBS for 24-48 h.

3.Digestion of cells with trypsin can harvest G0-phase cells.

3. Thymidine Double Block Method — Most commonly used. 

Day 0: Cell seeding (30-40% density).

Day 1: First thymidine block (2 mM, 16 h)

↓ PBS wash 3 times, change to fresh medium.

Day 2: Release culture (9 h)

↓

Second thymidine block (2 mM, 16 h)

↓ PBS wash 3 times.

Day 3: Synchronized cells obtained (at the G1/S boundary).

Samples can be taken every 2 hours to track the cycle progression.

Key points to note for floating cells:

I. Cell Washing:

1.Transfer the cell suspension to a centrifuge tube.

2.Centrifuge at room temperature or 37°C, 300×g, for 5 minutes.

3.Discard the supernatant and gently shake the tube to disperse the cell clumps.

4.Add 37°C pre-warmed medium/PBS and gently resuspend by pipetting. 5. Repeat 2-3 times.

II. Anti-clumping Techniques:

-Use silica-coated centrifuge tubes.

-Add 0.1-0.5 mM EDTA (does not affect most cells).

-Use wide-bore pipette tips to reduce mechanical shear.