Cell Cycle Phases Not Resolved in DNA Histogram

Symptom

Histogram for DNA content does not show distinct G0/G1, S, and G2/M phase peaks. Peaks are broad with high coefficients of variation (CVs).

1. 1 Flow rate set too high on cytometer, leading to high CVs and loss of phase resolution
2. 2 Insufficient staining with Propidium Iodide/RNase solution (CST #4087) or inadequate incubation time < 10 min
3. 3 Cells harvested during non-exponential or synchronized growth, missing representation of all cell cycle phases

1. 1 Run samples at lowest flow rate setting on cytometer to minimize CVs and maximize phase resolution
2. 2 Resuspend cell pellet directly in PI/RNase solution (#4087) and incubate for at least 10 min; use alternate DNA dyes DRAQ5 (#4084) or DAPI (#4083) if PI channel conflicts exist
3. 3 Harvest cells during asynchronous and exponential growth to ensure all cell cycle phases are accurately represented

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