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Flow Cytometry (CST Guide) moderate

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).
Common Causes
  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
Solutions
  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
Related Video (3)
Bilibili (China-Accessible Mirrors) ★ 85
Ten-Minute Guide to Flow Cytometer Instrument Operation
"Directly demonstrates flow cytometer parameter settings including histograms, which is where the high flow rate causing broad peaks occurs."
Bilibili (China-Accessible Mirrors) ★ 75
BD FACSCelesta Software Operation Training
"Hands-on instrument setup and data acquisition training shows the operational controls relevant to adjusting flow rate during acquisition."
Bilibili (China-Accessible Mirrors) ★ 70
Flow Cytometry Complete Workflow: Sample to Analysis
"Full workflow with troubleshooting covers the acquisition-to-analysis path where poor phase resolution would be diagnosed and corrected."
Source: cellsignal.com ↗
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