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Compensation

There are a wide variety of commercially available fluorochromes conjugated and unconjugated to monoclonal antibody reagents which are used to detect a wide variety of surface antigens. The choice of fluorochromes is limited by the laser excitation sources available in your flow cytometer(s). The instruments available at the Blizard core facility allow most of the commercially available fluorochromes to be used with some limitations.

Each fluorophore emission spills over into each other and this needs to be electronically removed or compensated from the other fluorophores in the experiment. This process requires the investigator to prepare cells labelled with fluorophores separately to allow the compensation process to be carried out precisely. The compensation process works better if Becton Dickinson compensation beads are used as the automated FACSDiva software algorithm has to reference non-fluorescent material, thus the use of unstained cells will give incorrect compensation values, which can be corrected manually. These beads provide the researcher with a physically separate positive and negative peak (non-fluorescent material) for each fluorochromes used. This is particularly useful and necessary when using antibodies for low expressing antigens (e.g. cytokines) which do not show a clearly separate fluorescent peak.

The compensation process can be done in an automated or manual manner within the FACSDiva software. The automated compensation program is recommended as the software automatically checks the effects of each change in compensation made on all other parameters used something difficult or very time consuming when carrying out the compensation procedure manually. If using the automated program users should check the compensation control compensation matrix.

The use of 'compensation beads' has several benefits, firstly the user does not repeatedly have re-run single colour controls and the beads give distinct positive and negative peaks which is not always possible given variations in antigen expressions. The positive bead peak has to be higher on the fluorescent scale than the cells employed, if the cells are lower the compensation is valid. Cell samples stained with a single colour will be over compensated when the whole compensation matrix is applied. The correct compensation for single cell colour controls is the compensation covering that particular fluorochromes bleed through to the other fluorochrome channels used. If using isotype controls there fluorescence intensity can be compared to the test antibody by binding the isotype to comp beads. This can help in determining the efficacy of use of such isotype antibodies.

The automated instrument tracking software or BD Cytometer Setup and Tracking also adjusts voltages on PMTs so that every preset compensation matrix will be valid if the save settings option is chooses within the software, see tutorials below for more detailed information.

  • Spectral Overlap
  • Manual FITC-PE compensation tutorial
  • BD FACS Diva software automated compensation tutorial
  • Anti-rat BD compensation bead protocol
  • Anti-mouse BD compensation bead protocol

Short list of fluorochromes, cell dyes & stains available

  • Antibody fluorochromes, cell dyes & stains

Antibody fluorochromes and DNA dyes used on Blizard Flow Cytometers

  • BD FACSAria III
  • BD LSRII
  • BD FACSCanto II
  • BD FACScan
  • Muse

Quantum dots can also be used on the flow cytometers at The Blizard Institute, these include Qdot 525, Qdot 585, Qdot 605, Qdot 655, for details about the use of Qdots please go to the section on Quantum Dots.

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