In the field of biotechnology and biomedical research, the flow cytometer has established itself as an essential tool. It allows detailed analysis of cells and particles in suspension. This technique simultaneously assesses multiple cellular characteristics, such as size, internal complexity and surface markers. It is therefore indispensable in disease research, diagnostics and cell therapy. In particular, flow cytometry for exosome characterisation allows the analysis of these extracellular vesicles. This provides valuable insights into their role in various biological processes and diseases.
The following describes the key steps in working with a flow cytometer, with a focus on optimising results and ensuring accurate data. This process is crucial for highly complex research, such as those involving exosomes or any other analysis related to advanced cell biology.
Proper sample preparation is essential to obtain accurate results in the flow cytometer. The first step is to isolate and suspend the cells or particles in a suitable buffer that prevents agglutination and allows optimal flowability. In the case of exosome characterisation, it is necessary to isolate the exosomes first, which involves techniques such as ultracentrifugation or specific kits such as those from Immunostep that allow the precise capture of exosomes using specific antibodies.
Samples should be filtered to remove unwanted cell debris or fragments that may interfere with the measurement. For exosomes, it is recommended to use reference nanoparticles to establish size and concentration controls.
Depending on the objective of the research, it is crucial to select specific antibodies that will mark the cellular components of interest. In the case of exosomes, antibodies targeting specific membrane proteins, such as CD9, CD63, and CD81, which are classical exosome markers, should be chosen.
The use of fluorescent markers is key in this step, as the flow cytometer uses laser light excitation to measure fluorescence intensity and correlate it with the presence of a specific marker on cells or particles.
Before starting the analysis, the cytometer must be properly calibrated. This includes adjusting the lasers according to the wavelength of the fluorochromes used, as well as setting up the detection channels to record the fluorescence signals accurately.
At Immunostep, they excel in offering advanced solutions in flow cytometry technology, including reagents and calibration kits that ensure optimal equipment performance. Positive and negative controls must also be incorporated into this process to validate that the data obtained is reliable.
With the samples prepared and the cytometer calibrated, it is time to acquire the data. During the analysis process, the flow cytometer passes the sample through a laminar flow and illuminates it with a laser. Particles (cells or exosomes) scatter light, allowing detection of characteristics such as size and internal complexity.
In addition, fluorochromes associated with specific antibodies emit fluorescent light, which is captured by the cytometer detectors.
Once the data have been acquired, they are analysed and processed. This step is key, as it allows the signals obtained to be interpreted and the characteristics of the cells or exosomes to be determined. In this phase, flow cytometry software plays a crucial role, allowing the identification of cell subpopulations. In addition, it facilitates complexity analysis and accurate quantification of marker expression.
It is important to perform data quality control to ensure that signals are not contaminated by noise or artefacts. Often in exosome studies, this includes verification of sample size distributions and purity, which must meet established parameters.
Finally, researchers must interpret the results in the context of the research objective. For example, if they are assessing the interaction of exosomes with target cells, they should determine how many exosomes are positive for a specific marker. They should then compare these data with controls.
It is also advisable to include relevant statistics, such as mean, median and standard deviation. These data support the robustness of the results.
The final report should be clear and detailed. In addition, it should include representative graphs, such as scatter plots or contour plots, to facilitate visual interpretation.
Working with flow cytometers is a process that requires detailed technical knowledge and a rigorous approach to ensure accurate results.
From sample preparation to the final interpretation of the data, each step is crucial to obtain valuable information. This information focuses on cellular characteristics and their interactions. In this regard, Immunostep’s advanced solutions offer specialised tools to optimise the analysis of exosomes and other biomarkers. These tools increase the accuracy and reliability of biotechnology studies.
By mastering each of these steps, researchers can obtain more robust and meaningful data. This opens the door to new discoveries and breakthroughs in biomedicine and cellular therapies. If you would like more information, please contact us: info@immunostep.com