Flow cytometry (CMF) is a technique based on the use of laser light, very useful for counting and studying cells and their characteristics.
To correctly perform flow cytometry and obtain the best possible results in your research, it is important to take several aspects into account.
One of the most important keys to properly perform flow cytometry is sample preparation. From Abyntek, we have some tips to keep in mind when preparing your sample:
CHOOSE THE RIGHT TEST TUBES
Depending on the cytometer you have in your laboratory, you will need to use one type or another of test tube: with round bottom or flat bottom, made of polystyrene or polypropylene, etc.
This prior verification will allow you not to be transferring your sample from one tube to another and, therefore, the possibility of losing part of the sample in these transfers, which could affect the number of initial cells.
Furthermore, depending on the type of plastic from which the tube is formed, the cells will adhere to it with more or less affinity. For example, cells like monocytes adhere more strongly to surfaces like polystyrene and less strongly to polypropylene.
So, if you take these aspects of the test tubes into account, you can better preserve your sample.
PRESERVE THE CONDITIONS OF THE SAMPLE
It is important, in this sense, not only to preserve the sample in the appropriate conditions, but to be especially cautious in the different processes that are carried out in your experiment. For example, in the centrifugation, in the agitation of the sample (it is convenient to avoid the use of the vortex) or in the aspiration of the supernatant.
PREVENT CELL AGGLOMERATION
To correctly perform flow cytometry, the laser must detect the cells individually, so it is important to try to avoid, as much as possible, the agglomeration of the cells.
To avoid this agglomeration, apoptosis and / or cell necrosis must be prevented, specific buffers must be used to prevent the formation of extracellular traps, the DNA released by dead cells must be eliminated by using DNAses, or the adhesion of cation-dependent cells must be avoided using EDTA.
DELETE NON-SPECIFIC JOINTS
To obtain good results in our flow cytometry, it is essential that nonspecific binding of the antibodies to the cells of the sample does not occur. In order to avoid this union, blocking solutions can be used. Another option is to incubate the sample with serum of the same species as the antibodies to be used.
Eliminating this type of junctions is especially important if the analysis that you are going to do focuses on the use of monocytes or dendritic cells, since this type of cells can express a range of Fc receptors that have the ability to bind antibodies through their Fc region, rather than through its antigen-specific binding site.
ELIMINATE DEAD CELLS
The presence of dead cells in the sample can lead to false positives in our flow cytometry, since they generate a certain degree of autofluorescence.
Some common methods to remove them are using FSC or SSC. The use of viability markers is also recommended to help identify these dead cells.
So, taking into account these tips to try to keep your sample in the best conditions, you can ensure that your experiment has the best possible start, and thus be able to correctly perform flow cytometry.