HomeThere 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 ICMS 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. These beads provide the researcher with a physically separate positive and negative peak 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 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 and DNA dyes used on ICMS Flow Cytometers
Quantum dots can also be used on the flow cytometers at ICMS, 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.