Flow Cytometry Cell Surface Staining Protocol

Materials Needed

• Cell sample (fresh or previously frozen)
• Blocking buffer (e.g., PBS + 10% FBS)
• Fluorochrome-conjugated antibodies
• 96-well V-bottom plate or tubes
• Ice bucket
• Centrifuge
• Flow cytometer

Protocol

1. Cell Collection and Preparation
   • Collect cells and adjust the concentration to approximately 1x10⁶ cells/mL in phosphate-buffered saline (PBS).
   • Centrifuge at 300 g for 5 minutes and aspirate the supernatant.
2. Blocking
   • Resuspend cells in 100 μL of blocking buffer (e.g., PBS with 10% fetal bovine serum and/or other commercial blocking reagents) to reduce non-specific antibody binding.
   • Incubate for 10 minutes at room temperature.
3. Staining
   • Add the antibody as recommended by the manufacturer, to the cell suspension.
   • Incubate for 30 minutes at 4°C in the dark to prevent fluorochrome degradation.
4. Washing
   • Add 1-2 mL of PBS and centrifuge at 300 g for 5 minutes.
   • Aspirate the supernatant and resuspend the pellet in 500 μL of PBS.
5. Acquisition
   • Analyze on a flow cytometer. Adjust the voltage settings to ensure the fluorescent peaks are on scale and clearly distinguishable.
6. Data Analysis
   • Use software to analyze the data, setting gates based on the controls to identify positive populations.

Important Considerations

Sample Preparation

• Always keep cells cold (4°C) throughout the procedure
• Use viability dyes to exclude dead cells
• Include unstained and single-stain controls
• Consider Fc receptor blocking for certain cell types

Antibody Selection

• Select antibodies specific to the cell markers of interest and compatible with the species of your sample
• Choose fluorochromes that do not have overlapping emission spectra to avoid signal overlap, which can complicate analysis
• Include appropriate controls such as unstained cells, single-stained controls, and fluorescence minus one (FMO) controls to accurately set gates and interpret data

Tips and Tricks

1. Optimization
   • Always titrate new antibodies
   • Run voltage optimization on your instrument
   • Use compensation beads for setting up compensation
2. Quality Control
   • Filter samples before acquisition to remove clumps
   • Include counting beads for absolute counts
   • Run quality control beads regularly
3. Troubleshooting
   • High background: Check for proper washing or add blocking agents
   • Poor separation: Verify antibody titration, or reduce background by adding blocking agents
   • Low event rate: Check for cell clumping, dead cells, or improper washing
4. Best Practices
   • Prepare fresh buffers regularly
   • Protect fluorochromes from light
   • Document all experimental details

Safety Notes

• Wear appropriate PPE
• Handle human samples in BSL-2 facilities
• Proper disposal of biohazardous materials
• Use caution with sodium azide-containing buffers