Preparation

• Wash cells twice with PBS + 2% FBS
• Resuspend with 100ul of PBS + 2% FBS
• Take down to BD S8

Running Samples using BD S8 FACSChorus

1. Select fluorescence markers
   • Draq5
   • Alexa Fluor 488 Beta-catenin
2. Select image channels depending on the fluorescence marker
   • Draq5 was in Imaging channel 3
   • Beta-catenin was in Imaging channel 1
3. Run the full stain to adjust gains
   • Spectral unmixing [when saturation is too high]
4. Backflush to get rid of any cells in the flow.
5. Run single samples [MAKE SURE TO RECORD THESE]
   • Unstained first [set this as the negative control]
   • Rest of the single stains [So Draq5 separately, Beta catenin separately]
6. Run full stain [View Data]
   • Add histograms & extra plots you want
     • Here we did a histogram with Image Channel 1 vs Image Channel 3 as a log histogram
     • Expecting double positive cells in the top right corner
   • Use distribution plot to view cells in the cell cycle
   • Select the peak on the right hand side as the “end flow”
     • This means once there is x events [cells] that are in that region then it stops recording

<aside> 📌 note! flow rate = how fast cells flow through [don’t want this too high or we might get doublets and unclear images!

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