Advice on compensation controls

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Because flow cytometry uses multiple fluorophores with overlapping emission spectra, 'compensation' is required to separate these signals. To perform compensation, a set of single colour controls (or compensation controls) are required. One single-stained control for each fluorophore being used in the experiment is required, along with a negative signal for each colour (either in the form of a negative peak in each single colour control, or less desirably, a separate negative control tube).

Requirements for correct compensation:

  • Comp sample must be stained with only one colour (must have a 'positive' signal, and where possible should include a 'negative' signal as well)

  • Positive signal in each control must be at least as bright as sample

  • Must have sufficient numbers of positive and negative events for statistical calculations

  • Positive and negative populations must have same autofluorescence

  • Signals must be on scale AND in the Linear Range

  • Compensation samples must treated in exactly the same manner as expt samples (fix/perm/incubations etc)

  • Verify compensation using a display transformation (“biexponential display”)

Laboratory practices:

  1. Use compensation (COMP) beads where possible instead of cells

  2. Use lot-matched negative and positive compensation beads

  3. Stain both negative and positive COMP beads in the same tube with a single fluorophore-conjugated antibody

  4. Treat your COMP beads the same as your samples: every step from antibody staining afterwards, especially fixation and washing

  5. Where possible, use the exact same antibody tube for your COMP beads and sample

  6. Typically, use fixed COMP beads/samples within three days

  7. Ensure your voltages are set so that your signals are on scale

Notes on compensation beads:

  • Generally brighter than cells... but not always (CHECK)

  • Make sure the negatives have the same autofluorescence as the positive

  • articular problem with violet channels

  • se “unstained” capture beads rather than “negative” beads

  • Not recommended for V500/AmCyan Reagents (“spectral differences”??)

  • Increased autofluorescence in the 450nm range (cells and possibly beads)

Computer/FlowJo practices:

  1. Draw the desired “single bead” or “single cell” gate yourself

  2. Use “negative” and “positive” gates from the same sample (as opposed to using a universal negative)

  3. Draw “negative” and “positive” gates yourself if the distinction is not clear – sometimes FlowJo makes a mistake

  4. Check a fully stained sample on an NxN plot – look for tell tale signs of bad compensation