Mike Barnkob:Protocols/Immunology/Flow/General notes

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General notes on flow cytometry

Antibody selection

  • 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

Isotype controls

  • 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

Tamdem dyes

  • 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

Compensation

  • 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.

Doublet exclusion

  • 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

Sorting

  • 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
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