Immunophenotyping

Multiparametric analyses

The most powerful aspect of flow cytometry is the simultaneous analysis of multiple properties of single cells. Our LSR Fortessa cytometer is capable of analysing signals of up to 13 fluorochromes at the same time. It is becoming routine to do 5 to 9 colour experiments without major difficulties. There are few basic rules that one should consider when choosing a combination of fluorochromes:

  • Check on our website in the Equipment section (Configuration of lasers) what fluorochromes we can excite and detect.
  • Choose the brightest fluorochrome for the least abundant antigen and the dimmest fluorochrome for the most abundant antigen.
  • Select fluorochromes taking into consideration the minimalisation of spectral overlap. Each fluorochrome has a wide fluorescence emission spectrum extending beyond the narrow window of light passed by an optical filter for a specific fluorochrome. Highly overlaping signals are difficult to compensate. There are spectra viewers (BD, ThermoFisher) available that can help you to make the choice, whether two or more fluorochromes can be combined together or not. You can always count on our help in this regard.
  • Check the availability of antibodies conjugated to the desired fluorochromes.


Controls

To make proper experimental setting and draw appropriate conclusions about your samples the negative and positive controls are necessary.

Negative controls: Unstained cells.

Compensation controls: In multiparametric experiments, it is necessary to prepare controls stained with each fluorochrome separately. These single color controls are required for compensation of signals between detectors and are crucial for multicolor immunophenotyping. Single stain controls can be performed on compensation beads or cells. For compensation purpose it is crucial that the single stained conntrol is POSITIVE for the antigen of interest. If you are not sure, whether your cells produce the chosen protein, use beads or other cells that are positive for the antigen. In both cases you also need the unstained samples (beads or cells).

Specificity controls: In case of indirect antibody staining, meaning the use of primary and secondary antibodies, it is important to include a control stained with the secondary antibody only.