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Flow Cytometry with beads

Updated 7/6/2016 12:08 AM
Step-by-step
by Hannah Shen (created by Sebastian M. Castillo Hair)

Introduction

Written by Sebastián Castillo Hair from Jeffrey Tabor's lab at Rice University. Note: this procedure is referential only. Specific details may vary depending on your instrument, software, or beads manufacturer.

Materials

  • Flow cytometer
    • Calibration beads
      • Cell samples to be measured
        • Phosphate Buffered Saline (PBS) or sheath fluid

          Procedure

          1. Initialization

          1. Check levels of sheath/waste fluid and replace as necessary.
          1. Turn the flow cytometer on.
          1. Run any required initialization procedure: fluidics startup, etc.
          1. Open acquisition software.

          2. Measure calibration beads

          1. Prepare a flow cytometer tube with ~250µL PBS.
          1. Vortex vial containing calibration beads.
          1. Add one drop from the calibration beads vial into the flow cytometer tube.
          1. Vortex flow cytometer tube.
          1. Insert tube in the flow cytometer.
          1. Tune the Forward Scatter/Side Scatter gain and threshold settings so that beads events appear in the center of the Forward/Side Scatter plot.
          1. If possible, tune the flow rate settings so that between 500 and 1000 events per second are registered.
          1. Tune the relevant fluorescence channel settings so they match what will be used for acquiring cell data.
          1. Acquire data and save into a .fcs file. Acquiring around 30000 events is recommended.
          1. If different acquisition settings will be used for different cell samples, repeat steps 2.12 and 2.13 for each different set of settings.
          1. Store vial of calibration beads at 4°C.

          3. Measure cell samples. For each cell sample:

          1. Prepare a flow cytometer tube with the desired amount of PBS.
          1. Add the desired amount of cell sample to the flow cytometer tube.
          1. Briefly vortex the flow cytometer tube at a slow speed.
          1. Tune the Forward Scatter/Side Scatter gains and thresholds as necessary.
          1. Tune the relevant fluorescence channel settings as appropriate. Record which beads .fcs file matches the acquisition settings for the current cell sample.
          1. Acquire data and save into a .fcs file. Acquiring at least 25000 events is recommended.

          4. Flow Cytometry Shutdown

          1. Save your new .fcs files.
          1. Perform any required shutdown procedure (bleach wash, fluidics shutdown, etc).
          1. Close acquisition software.
          1. Turn the flow cytometer off.

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