The quality and consistency of the antibodies are essential aspects if you want to obtain reproducible results in scientific research. And these aspects become especially critical in the case of using antibodies for Flow Cytometry .
In this entry we discuss 3 key aspects that need to be considered to ensure that the Flow Cytometry antibodies work as expected.
1.- Titration
Why is it important to titrate antibodies for Flow Cytometry?
At high concentrations of antibody, the target proteins can become saturated causing the remaining antibody to bind with low affinity to other antigens present in the cells.
This fact would lead to an increase in background noise, increasing the fluorescence due to non-specific junctions.
To avoid such out-of-range junctions, it is essential to titrate the antibodies before proceeding to the flow cytometric assay. This minimizes noise due to nonspecific binding of antibodies to low affinity antigens.
Ultimately, it’s about finding the antibody concentrate that offers the brightest signal along with the least background noise.
How to titrate the antibodies for Flow Cytometry?
Antibody titration for Flow Cytometry should be done under the same conditions under which the assay will be conducted, but with varying concentrations of antibody.
Typically, a series of 6-8 serial dilutions of the antibody will be performed and tested against a known number of cells. With these data, a standard curve is constructed to determine the concentration at which a better signal-to-noise ratio is obtained.
In this post we tell you in more detail how to titrate the antibodies.
2.- Specificity
Why is it important to determine the specificity of antibodies for Flow Cytometry?
The specificity of each antibody must be validated for each specific technique, since the same antibody could work perfectly in an ELISA immunoassay and yet not yield the expected results in Flow Cytometry.
In this post we expand the information with some tips to validate antibodies.
How to determine the specificity of the antibodies for Flow Cytometry?
In the case of Flow Cytometry, the validation of the specificity can be carried out through different routes such as, for example, the inspection of the cells under the microscope once they have been marked with the antibody, or the development of positive and negative cell lines using CRISPR techniques or siRNA.
3.- Controls
Why is it important to use controls in Flow Cytometry?
The use of controls (especially during the panel development phase) is imperative to ensure that both the protocol and the reagents are working as they should.
What controls should be used in Flow Cytometry?
Some of the recommended controls for Flow Cytometry are the following:
- Cell viability control
The presence of dead cells in the sample can lead to artifacts due to nonspecific binding of the antibody, thereby increasing autoflorescence levels.
There are markers (7-AAD, Nuclear Green DCS1, propidium iodide, etc.) that discriminate between living and dead cells, staining only the latter.
- Spectral Overlay Control
By performing a multi-color Flow Cytometry assay, the emission spectrum of various fluorochromes could overlap.
This fact can be corrected by means of a compensation process, by means of which said spectral overlap is estimated, and then subtracted from the total detected signal.
- Autofluorescence control
Some cellular components (for example NADPH or flavins) are capable of fluorescing by themselves, masking the specific signal of the antigen.
To detect the presence of autofluorescence in a sample, an unmarked aliquot was previously analyzed by Flow Cytometry.
Autofluorescence can be corrected by using a different laser, or as in the previous case, by compensation.
- Control of unwanted antibody binding
The antibody could bind to out of range epitopes present in the sample that do not constitute the target of interest.
To correct it, it is always advisable to include:
– Negative controls (cells that do not express the antigen of interest)
– Isotype controls (antibody of the same isotype as the primary one, but specific to an antigen not present in the sample)
– Isoclonic controls (unlabeled antibody)