Autofluorescence Controls...

Are most appropriate when your samples may exhibit naturally high levels of autofluorescence that may differentially impact the sensitivity of resolution in each detection channel.

Different cell types and tissues have varying levels of inherent fluorescence. Major sources of autofluorescence include NADH, riboflavin, metabolic cofactors, the crosslinking of primary amines by paraformaldehyde, and certain biological structures (e.g., mitochondria, lysosomes). These proteins and molecules are more easily excited at lower wavelengths (i.e. from the UV, violet, and blue lasers) and will emit at a wide range of 300-600 nm, which overlaps with several common fluors like BV421™, Pacific Blue, and FITC. Myeloid cell lineages tend to be particularly autofluorescent. Furthermore, stimulating cells can cause them to become more metabolically active, producing more autofluorescent proteins, vitamins, and cofactors. An unstained control sample is helpful in delineating how much autofluorescence populates your channel of interest. Also, autofluorescence doesn’t have a large Stokes shift (the difference in nanometers between the peak excitation and emission wavelengths), so tandem fluorophores can prove very useful to resolve populations on highly autofluorescent cell samples. If necessary, these factors can be taken into consideration when building a flow panel.

Stimulation Controls...

Are most appropriate when you have samples that are stimulated or exogenously treated.

Background signal can change when you stimulate samples, potentially affecting population resolution. For example, PMA stimulation can cause a decrease in surface CD4 expression in T cells while also increasing the level of autofluorescence in short wavelength emission channels with the production of autofluorescent molecules. Alternative gating strategies may have to be used (i.e., CD3+, CD8-, CD56- events) or the intracellular detection of CD4.



Human PBMCs were either untreated (left) or stimulated with 
PMA/Ionomycin for six hours (right). Samples were analyzed two days later.

In other cases, it helps to compare stimulated samples to the basal level of expression in unstimulated controls. Unstimulated controls help to establish where gates should be drawn and also let you better analyze the amount of meaningful upregulation of your target.



Human PBMCs were either untreated and stained with the full panel (orange), or stimulated with 
PMA/Ionomycin for six hours and stained with the full antibody panel (red) or an FMO for IFNγ (blue). 


Human Variability Controls...

Are most appropriate when you want to identify human-to-human variation.

There's a large amount of genetic variation from human to human, and this can account for some unexpected differences in expression of certain markers. This is best demonstrated in patient samples, where antigen expression, autofluorescence, and background variance often require, for the best results and accurate gating, that each fully-stained patient sample has controls derived from the same patient. Alternatively, you might consider using Veri-Cells™, a normal human control sample that can verify the consistency of reagent performance between experiments.

In the data below, two human donors display consider variability in their expression of CCR4 and CCR5. On NK cells, Donor B exhibits very low CCR4 levels, but higher levels of CCR5 when compared to donor A. In this scenario, the reagents may not be at fault. Rather, human genetic variability may explain the observed differences in staining. 


Live/Dead Controls...

Are most appropriate when you want to exclude dead cells and debris from your analysis.

Since dead cells and debris can non-specifically stain with antibodies and can also have antigen expression that is not consistent with live cells, it is helpful to exclude them from analysis. DNA stains like Propidium IodideHelix NP™ NIR, and Helix NP™ Green are cell membrane impermeant, meaning they only enter cells with compromised membranes, such as dead cells. For cells that will otherwise be analyzed unfixed, nucleic acid/DNA stains are the preferred live/dead indicator.

For samples that need to be fixed and permeabilized (particularly with ethanol or methanol), DNA binding dyes are often not ideal. This is because the DNA can be denatured, thereby releasing intercalated dyes and allowing them to non-specifically bind cells that are now fixed, permeabilized and thus “dead”. In addition, cells that were originally dead may lose their signal amplitude as the dyes escape. For these experiments, we highly recommend Zombie dyes, which covalently attach to primary amines instead of DNA. If the cell is alive when labeled, only amines at the cell surface will be bound. If the cells are dead when labeled, the amplitude of the signal will be several folds higher since the dye now has access to intracellular, amine-containing proteins. Zombie dyes are helpful for studies involving cytokine and transcription factor analysis requiring fixation and permeabilization.


One day old C57BL/6 mouse splenocytes were stained with Zombie Red™ and analyzed before fixation (purple) or after fixation and permeabilization (red). Cells alone, without Zombie Red™ staining, are indicated in black.