05 Preparing Controls

2026-02-11

Recommendations for controls

Here are my recommendations for preparing the controls. These are not substantially different from the recommendations from Mario Roederer over twenty years ago, or those from [Laura Ferrer-Font](https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpz1.222. One aspect that I would emphasize, is to take care in your panel design to avoid generating situations where it is difficult to produce good controls. One key area is with tandem dyes. Since tandem dyes are particularly prone to mismatch between the controls and the fully stained sample, minimize the total amount of signal from tandems in your panel to minimize the total error. By this, I mean things like not putting PE-Cy7 on CD45RA.

• Use cells whenever it’s easy (e.g., CD4, CD14)
• Use cells if the marker is highly expressed (bright)
• Use cells for the viability stain
• Use beads if the marker is rare and low expression
• Try to avoid rare + very bright in panel design
• Use simple fluorophores (e.g., PE, or single peak)
• Try to avoid rare + tandem in panel design
• Avoid high expression + labile tandem in panel design
• Ensure that your controls are treated the same as your samples
• Match the autofluorescent background between positives and negatives
• For lymphocytes, use a negative set on the lymphocyte gate
• For monocytes, use a negative set on the monocyte area (e.g., separate unstained)
• Use the same protocol and buffers EXCEPT: no Brilliant Stain buffer
• The controls need to be as bright as the brightest events in your fully stained samples. The separation between positive and negative in your controls is important, which is one reason why having a universal (cold) negative helps.
• Use tandem stabilizer from BioLegend throughout