How Many Sample Tubes Should be Prepared for a Flow Cytometry Experiment and How to Setup the Control?

Do you have this question in mind when doing a flow cytometry experiment: How many controls do you need and how many sample tubes should you prepare for the control setup in a complete flow cytometry experiment?

Elabscience® summarizes 6 common flow control setups for you and provides sample tube setup references using the example of a three-color experiment with CD3-APC, CD4-FITC, and CD8-PE.

01 Blank Control - Used to Adjust Instrument Voltage

Blank control refers to the control tube without any fluorescent dye added. The blank control is set up to differentiate between the inherent fluorescence of the cells and to determine whether external factors such as drug treatment may introduce additional spontaneous fluorescence.

In addition, the cell population in the blank tube is the most possible negative population, which can assist in the adjustment of voltage and avoid the line compression of negative cell populations due to low voltage during instrument operation.

02 Single Positive Control - Used for Multi-color Compensation Adjustment

A single positive control is a sample tube that only contains one fluorescent antibody. In a multi-color flow experiment, due to the wide coverage of the emission wavelength of the fluorphore, fluorescence may overlap and cause some interference with the data analysis of the experiment results. In this case, we usually set up a single positive tube for compensation adjustment. At the same time, the single positive tube can also assist us in adjusting the channel voltage to prevent the signal from exceeding the receiving range.

Typically, you need to set up as many single positive tubes as there are fluorescents in the experimental panel.

03 Isotype Control - for Setting Gates

Isotype control refers to an antibody that has the same species origin, subtype, fluorescence label, dose and concentration as the antibody used, but does not bind specifically to the target site. Isotype controls are commonly used to eliminate non-specific binding of antibodies causing background staining.

04 FMO Control - for Setting Gates

FMO control is an abbreviation for Fluorescence Minus One. In multi-color experiments, it may be difficult to distinguish signals in some channels, mainly because another fluorescence interferes greatly with that channel, and it is not possible to use a blank control or all isotype controls to set gates. Remember that as you add more fluorescences, overall fluorescence leakage will change, so you need to use fluorescence compensation controls (i.e. remove one of all fluorescence labels).

05 FMO Combined Isotype Control - Used for Gatesetting

The FMO combined isotype control refers to the combination of FMO and the isotype control antibody corresponding to the antibody that has been removed. FMO control and isotype control reflect two types of background fluorescence, so using one of them alone to gateset in multicolor flow experiments is not reliable. Currently, people are trying to combine FMO (considering fluorescence spillover) and isotype control (considering non-specific antibody binding) to gateset, which is also a relatively easily accepted control for journal submissions.

06 Experimental Control - Used for Solving Research Problems

Experimental control, also called biological control, is not used for compensation or gatesetting, but for comparing differences between samples and reference standards under specific experimental conditions. For example, when studying the effects of a drug on a cell line, an untreated cell line of the same type must be set as the experimental control.

Example: A complete 3-color experiment sample tube set-up with CD3-APC, CD4-FITC, and CD8-PE:

Note: "-" means no antibody added, "*" means adding the corresponding isotypic control.

Explanation:

1. Generally, indicators such as CD3, CD4, CD8 that have clear grouping do not need to set isotype controls;

2. Whether to set FMO control depends on the degree of fluorescence interference. In this experiment, FMO joint isotype controls are not required;

3. For panels with low fluorescence interference (such as in this experiment), it is recommended to do a single positive control for the first experiment, and no single positive control is required for subsequent experiments. If you are a new experimenter, it is recommended to do a single positive tube every time.