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Choosing a wrong antibody for flow cytometry can result in disastrous consequences for your project such as loss of time and of precious sample. To avoid the risk of choosing a bad antibody, ProteoGenix developed a hybridoma development service guaranteed for flow cytometry applications. How do we limit the risk?
We manage the whole process from the beginning to the end
We test the antibodies produced in your OWN conditions.
We send you part of the sample so that you can test it by YOURSELF in your own conditions.
You will be fully charged only if it worked when you tested it! TEST IT, VALIDATE IT, BUY IT! That’s our guarantee!
Polyclonal antibodies are generated by immunizing a host with your target antigen and by collecting the serum. It is the cheapest way to produce a large amount of antibodies against a given target. If your main concern is to detect a specific antigen on your cells, polyclonal antibodies could be enough. However, keep in mind that polyclonal antibodies suffer from low specificity compared to monoclonal antibodies leading to possible unspecific binding. This issue can be partially overcome by a careful antigen design.
Monoclonal antibodies are usually obtained by fusing mAb producing splenocytes with myeloma cells to form hybridomas. Hybridomas have the particularity to secrete an antibody identical to the one of the parent cell. Compared to polyclonal antibodies, monoclonal antibodies allow obtaining cleaner data thanks to their capacity to bind to only a unique epitope.
Any doubt about the best choice for your project? Contact our PhD account managers who will be happy to assist you!
One of the most commonly asked question when developing flow cytometry antibodies is “How should I choose between direct and indirect staining?”. Unfortunately, there is no clear answer to this question. However, we can try to share some elements that should help make a decision.
Direct staining refers to the use of a labeled primary antibody. This indicates that the light emitted by the fluorophore is directly proportional to the amount of antibody present on the cell (or in the cell in case of intracellular staining) and, thus to the amount of the target of interest.
In contrast to direct staining, indirect staining is based on the use of a labeled secondary antibody. Secondary antibodies are generally directed against antibodies from the host organism of the primary antibodies. This method presents two main advantages:
The primary antibody remains unmodified and, thus avoids the risks of potential sterical hindrance or specificity/function loss
It acts as a signal amplificator as several labeled secondary antibodies can bind to one primary antibody.
However, it should be noticed that the use of labeled secondary antibodies limits the possibilities of multiplexing. Indeed, as secondary antibodies are directed against the antibodies of a host organism, they will bind to each antibody generated from a same host. Concretely, two primary antibodies from a mouse could not be used in the same experiment because the contribution of each primary antibody will not be distinguishable.
To summarize, labeled primary antibodies will be preferred for experiments requesting measuring several parameters in parallel (multiplexing). Labeled secondary antibodies will be particularly useful for the detection of low abundance targets.
Flow cytometry is a commonly used technology for the characterization of single cells and particles. Basically, a flow cytometer is composed of several components:
Sheath fluid: the role of the sheath fluid is to focus the cell suspension through a small nozzle and to separate them. This separation process is particularly important to allow the laser beam to monitor each cell one at a time. A bad cell separation or a fast flow rate could result in a loss of information.
Laser: the laser illuminates each single cell with coherent light at a specific wavelength. Measurement of scattered light and fluorescence is the core principle of flow cytometry.
Detectors: detectors collect the scattered light and fluorescence. The optical signal is directed to the detectors thanks to a complex system composed of mirrors and filters.
In flow cytometry, cells can be distinguished depending on several parameters based on the measurement of scattered light and fluorescence:
The expression of a target protein
The two first parameters are measured thanks to the scattered light. Scattered light allows to differentiate various cell types by plotting scatter parameters (scattergram = forward angle light scatter vs. side scatter). Usually, the intensity of the forward light scatter is correlated with the cell volume while side scatter reflects the complexity of the internal cell structure.
Interestingly, cells can also be separated depending on whether or not they express a target protein. This can be achieved thanks to fluorescence-tagged antibodies which combine the optical properties of a fluorescent marker with the selectivity of an antibody. This allows the labeled antibody to bind to a single protein or modification and to serve as a label for the detection of cells containing the target. Please note that antibodies do not interfere with one another and that it is possible to detect multiple targets in parallel.
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