Looking for tips regarding isolating svfs and lymphocytes. In particular how to reduce the autofluorescence from lipids. Any suggestions would be greatly appreciated.
- *Use enzymatic digestion*: Enzymatic digestion can help break down adipose tissue and release cells, reducing autofluorescence caused by lipid droplets ¹.
## Use Appropriate Controls
- *Unstained cells*: Run unstained cells to determine morphological parameters and autofluorescence levels. This will help you set gates and compensate for autofluorescence.
- *Fluorescence Minus One (FMO) controls*: Use FMO controls to determine the background fluorescence and set gates for specific cell populations ².
## Select Suitable Fluorophores
- *Use tandem dyes*: Consider using tandem dyes, which can help reduce autofluorescence by shifting the emission spectrum.
## Additional Tips
- *Maintain cell viability*: Ensure cell viability is high to minimize autofluorescence caused by dead or dying cells.
- *Minimize exposure to light*: Keep cells in the dark as much as possible to reduce photoactivation of autofluorescent molecules.
By implementing these strategies, you can reduce autofluorescence and improve the accuracy of your flow cytometry results when working with adipose tissue.
We have been able to reduce autofluorescence drastically in our published protocol through filtration and digestion. Please refer to Delaney et al 2020 Analytical Biochemistry. Let me know if you need the full text.
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