Mike Barnkob:Protocols/Immunology/Flow/General notes
General notes on flow cytometry
- Selection bright fluorochrome (PE, brilliant violet, 488) for markers that are less expressed, and less-bright ones for markers that are highly expressed
- Auto-fluorescence is higher in lower light-ranges
- Use same species and IgG-isotype, with same fluorochrome is possible (meaning buying from same company as other antibody).
- Age affects fluorochrome
- Use at same concentration as target
- Unspecific binding: IgG1 < IgG2 < IgM
- Are affect more by temperature. Stain at 4c, not on ice. Also don’t keep in back of fridge, where it might be colder.
- De-conjugates more after dilution
- Beads are good, but: don’t account for fluorochrome changes and auto-fluorescence. Beads are best for T-cells, but work badly for many other cell types.
- Brilliant violet dyes are very hard to compensate, since they react with each other
- Tandem-dyes may uncouple
- During voltage changes – remember to check not only channel compensating, but also other channels which the color bleeds into.
- When seeing big data spread, consider 1) lowering voltage, 2) change antibody.
- Be especially carefully when doing CFSE staining, here it is better to take all.
- If set to stringently, will exclude cells that are dividing and of interest.
- FSC-A / FSC-W is better SSC-A/SSC-W.
- Consider doublet discrimination in the end, instead of after FSC-SSC
- Don’t use phenol red, increases auto-fluorescence
- Filter solution
- Wash at lower velocity (150g for 5 min)
- Sort into media
- Slowly shake the collection tube often, because cells will splash against walls of tubes and die if they don’t get into media