This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
, No Gclids
Experimental Controls Optimization
Selecting the Right FACS Controls
One of the first things to consider when designing your flow cytometry experiment is which controls to use. There are many sources for error and variability that can be introduced into experimental measurements, and it is important to control those points. The purposes of these controls are to ensure the flow cytometry instrument is functioning properly, that compensation is correctly set, that appropriate gates can be drawn, and the correct interpretation of the data can be obtained.
Use this table to help decide which controls are appropriate for your flow cytometry experiment:
| Control | What to include | Purpose | Notes |
|---|---|---|---|
| Unstained control | Unstained cells (incubated in parallel with your stained samples) that are fully processed without addition of any antibodies. | To control for background derived from auto-fluorescence, and to set the voltages and negative gates appropriately. Used as an additional negative control. | Comparison to beads can help to determine the relative amount of autofluorescence. Try using a different excitation source if autofluorescence levels are high. |
| Isotype control | Cells incubated with isotype control antibodies (antibodies usually raised against an antigen that should not be present in your cells). | To determine nonspecific binding of the primary antibody. | The isotype control should match: -The host species -Ig subclass (IgA, IgG, IgD, IgE, or IgM) of the primary or secondary antibody -Conjugated to same fluorophore as the primary antibody |
| Internal negative control | Population of cells that do not express the antigen of interest and are fully processed. | To avoid false positives resulting from nonspecific antibody binding. | Negative control cells are not always available. Ideally, the fluorescence intensity of the internal control should be the same as the unstained control. |
| Positive control | Cells known to express your target of interest. | To avoid false negatives resulting from a bad antibody. | Positive control cells are not always available. |
| Compensation controls | Compensation controls must match the exact experimental fluorochrome (with a similar brightness). The controls need to be at least as bright or brighter than any sample the compensation will be applied to, and background fluorescence should be the same for the positive and negative controls. | Allows you to remove (compensate for) spectral overlap. Spectral overlap should be compensated for every fluorophore used. | Single staining in multicolor flow cytometry is important due to spectral overlap between different fluorophores. Be sure to collect enough events to be statistically significant. |
| Viability control | Common viability dyes to identify dead cells include DNA dyes or protein binding dyes. | Exclusion of dead cells using viability staining means less non-specific binding and easier identification of positively stained populations. | Using a live/dead stain to remove dead cells can improve your staining. Dead cells have greater autofluorescence and increased non-specific antibody binding, leading to false positives and reducing the dynamic range. |
| Fc blocking control | Fc blocking reagents added to staining procedure; a commercial Fc block can be used. | To ensure that only antigen specific binding is observed (Antibody binding via Fc receptors can lead to false positives and data that cannot be interpreted). | Alternatively, the serum of the host primary antibody can also be used. For example, if your antibody is of a mouse isotype, you could use mouse serum to block non-specific binding of immunoglobulins/antibodies to the Fc receptors. For purified PBMCs, 10% human serum can be used. |
| Fluorescence minus one (FMO) controls | The experimental cells stained with all the fluorophores minus one for each fluorophore in your multicolor panel. | To detect influence of fluorescence spread within detection channels (especially with brighter fluorophores). | Important for building multicolor flow cytometry panels and helps determine where gates should be set. |
| Secondary antibody control | Cells incubated only with the secondary antibody. | To determine nonspecific binding of the secondary antibody. | Only necessary if using a secondary antibody. |
Flow Cytometry Technical Resources
Protocols, optimization tips, troubleshooting guides, and more for flow cytometry.
Technical ResourcesTroubleshooting Guides
Download troubleshooting handbooks for IHC, Western blot and ELISA for FREE.
Troubleshooting GuidesRelated Pages
Flow Cytometry Optimization Tips
Get some of the best Bosterbio's flow cytometry optimization tips. This guide includes protocols, optimization tips, troubleshooting tips, and more on flow cytometry.
See MoreFlow Cytometry (FACS) Troubleshooting Guide
Boster Bio is an antibody company and supplier. Read more about our troubleshooting tips for flow cytometry (FACS) experiments. Learn tips on how to resolve issues such as high background, and weak or no signal
See MoreFlow Cytometry Fundamental Principle, How FACS Works
Flow cytometry is a popular cell biology technique that utilizes laser-based technology to count, sort, and profile cells in a heterogeneous fluid mixture. FACS is an abbreviation for fluorescence-activated cell sorting, which is a flow cytometry technique that further adds a degree of functionality.
See MoreFlow Cytometry Technical Resource Center
Click Here for More Flow Cytometry Sample Preparation Every flow cytometry (or FACS) experiment begins with sample preparation. Click Here for More Flow Cytometry Technical Blogs Here is our ever growing archive of technical blog articles related to ...
See MoreFlow Cytometry Sample Preparation
Every flow cytometry (or FACS) experiment begins with sample preparation. Check out our flow cytometry sample preparation guide to learn how to prepare your samples for flow cytometric analysis.
See MoreFACS Flow Cytometry Technical Blogs
6 Helpful Tips FACS Multicolor Panel Design October 27, 2017 Keywords: flow cytometry, FACS antibodies, multicolor panels, CD antibodies, resources, fluorophores Planning to design multicolor panels for FACS antibodies? FACS Sorting Preparation Check...
See MoreAll Technical Support Resources
Boster provides comprehensive technical resources for applications, blogs, research area/pathway map /disease information, and web-based digital tools.
See MoreBoster Technical Blogs and News
FACS Sorting Preparation Checklist Mar 20 2017 Keywords: check list, technical tips, FACS, Flow cytometry, cell sorting preparation Are you preparing for a FACS experiment? 6 Helpful Tips FACS Multicolor Panel Design Oct 27 2017 Keywords: flow cytome...
See More