Immunophenotyping

Multiparametric analyses

The most powerful aspect of flow cytometry is the simultaneous analysis of multiple properties of single cells. It is becoming routine to do 5 to 9 colour experiments without major difficulties. Our LSR Fortessa cytometer is capable of analysing signals of up to 13 fluorochromes at the same time.

The detection of some fluorochrome combinations by conventional flow cytometry presents a challenge due to high amounts of spectral overlap. The Cytek Aurora, we are equiped in, addresses this challenge by using differences in full emission spectra signatures across all lasers to clearly resolve these combinations, even if the populations are co-expressed. With five lasers, three scattering channels (FSC, blue laser SSC and violet laser SSC) and 64 fluorescence channels, the Aurora suits every laboratory’s needs, from simple to highcomplexity applications. 40 colors demonstrated including fluorochromes with emission spectra in close proximity to each other.

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 (with our spectral cytometer - Cytek Aurora almost everything is possible) .

  • 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 or may generate substantial spreading. 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.