Storage and Handling
Make sure your antibodies are kept at 4°C (between 2°C and 8°C) and shielded from light exposure. Do not freeze antibodies conjugated to fluors, as this can cause precipitation or aggregation of the antibody. You should also make sure the product has not expired.
Are you using the recommended dosage per test? You may need to titrate your antibody to find the optimal amount to use for your unique experiment. Check your data sheet or CoA.
Be sure that your flow machine is equipped with the proper filters and lasers for the fluors you are interested in using. For example, Brilliant Violet™ 570 requires a violet laser and an optimal filter of 585/42. But, if someone tried to use the 525/50 filter they already have installed to detect BV570™, this would not work.
Fluors give off distinct emission profiles and the proper filters must be equipped in order to capture these wavelengths. In this instance, a 525/50 filter would catch wavelengths in the 500-550nm range. The peak emission of BV570™ (570nm) falls outside of this range and is not detectable by this filter.
Be aware of how many PMTs (Photomultiplier Tubes) each laser can handle. If you only have 3 PMT’s on the violet laser, but want to run 4 colors off it, this is not possible.
If the lasers in your flow cytometer are not well-aligned, this will cause a weak signal in some (or all) channels. It is easy to check for instrument performance using our Rainbow Calibration Particles. Beads will fluoresce in every channel, so if you don’t detect signal in one of the channels, this is an indication that the cytometer needs to be checked. Please follow the cytometer manufacturer's instructions for quality control checks and optimal performance.
Light can scatter from the 532nm laser going into the FITC filter off the 488nm laser. The same is true with scattered 561nm laser light into the PE filter off when using the 561 nm laser. Use of a notch filter in front of the filters for those PMTs can reduce the background caused by the laser light scatter, maintaining sensitivity in the PMT.
Certain markers are either expressed at low levels or on rare cell populations. In these instances, you should choose antibodies conjugated to the brightest available fluors:
Fluorophore Brightness Index
Certain haplotype-specific markers are only expressed by certain strains of mice, so carefully read the description before purchase. For example, H-2K, I-A, CD90, and CD45 for mouse, come in two or more characterized types, of which, each can be distinguished by haplotype-specific antibodies. In addition, as flow cytometry is based on hierarchal gating, an initial wrong turn in gate setting can have a negative downstream impact on data analysis. Be sure that you are examining the proper population before setting your initial gate. For example, if you are trying to examine Ly-6G on mouse bone marrow cells, it can readily be found within the granulocyte population, but not the lymphoid one. You may also want to make sure you’re examining the proper type of tissue/organ for the expression of your marker.
Be aware that some proteins can be expressed on the surface of the cell, in the cytoplasm, internal organelles, the nucleus, or in multiple compartments simultaneously. Be sure to follow the proper protocol for their detection.
Some markers are downregulated from the cell surface upon activation. Other markers and cytokines must be induced in order to be detectable by flow cytometry. The type, concentration, and duration of stimulation can all have an effect on how robust the expression levels can be. For cytokines, secretion should be blocked using Monensin or Brefeldin A. This prevents cytokines from being secreted into the environment.
Intracellular Cytokine Stimulation Guide
Are the proper buffers being used for your experiments? Our Foxp3 antibodies are optimized for use with our True-Nuclear™ Transcription Factor Buffer Set and may not work adequately with buffers from other vendors. In addition, certain flow cytometry applications require specific buffers for staining. The correct fixation and permeabilization buffers should also be used for intracellular cytokine staining. See our link below to examine our flow cytometry buffers in greater detail:
Flow Cytometry Buffers
If you are using purified or biotinylated antibodies, are the corresponding secondary antibodies correct? For example, if the primary antibody is an Rat Anti-Mouse, be sure to use a secondary reagent like Goat Anti-Rat. Using a tube with only the secondary antibody allows you to assess the level of background staining in your product. You may also want to test whether your secondary antibody is working with other primary antibodies. This would help determine whether the issue is with the primary or secondary antibody.
Are you fixing or permeabilizing your samples prior to staining? Fixation and permeabilization protocols can alter the interaction between epitope and antibody. This sensitivity is antigen-dependent and clone-dependent. For example, epitopes for clones like HIT2 (human CD38), 5C3 (human CD40), and BC96 (human CD25) are all particularly unstable with detergent or methanol treatments. In addition, some markers require a particular fixation for accessibility by antibodies, such as nuclear antigens. For instance, PCNA and Ki67 need ethanol fixation for its antibodies to bind properly. Your samples should never be exposed to fixation reagents for extended periods of time, as this can cause cross-linking which may affect the dye chemistry or increase autofluorescence.
Adherent Cells and Receptors
If you are working with adherent cells and use trypsin to detach them, be aware that trypsinization could potentially affect the expression of surface molecules. A similar phenomenon can occur if the cells are treated with collagenase prior to staining. If you experience a loss of signal, you may want to try a gentler method to retrieve your cells.
Receptors may be internalized during the staining. Always stain at 4°C and use ice-cold reagents, if possible. Receptors can also be internalized if the cells have been stimulated in vitro prior to staining. Adding sodium azide to your FACS buffer may help to prevent receptor internalization.
Noise and Background
Having high noise and background in your PMT might also be interpreted as low signal. This can be due to several factors such as scattered light from the laser source, spillover from other fluorophores, or excessive autofluorescence.
Spillover is the leading cause of loss of sensitivity in any particular PMT when performing multicolor flow cytometry. Appropriate compensation must be applied to eliminate the spillover signal from neighboring fluorophores or cross-beam excited fluorophores. If the percentages of that compensation are high, there is a good chance that sensitivity in that PMT will be reduced.