If you’re thinking about applying for a job that requires a flow cytometry interview, you need to be prepared to answer some challenging questions. Flow cytometry is a powerful tool used in medical and research laboratories, and employers will want to find out how well you understand and can apply the technique. By having a good understanding of the questions you may be asked, you can be confident in your answers and stand out from other candidates. This blog post will provide an overview of the types of questions you may encounter in a flow cytometry interview, helping you to prepare for the process and have the best chance of success. We will cover questions related to the principles of flow cytometry, the types of cells that can be analyzed, the use of flow cytometry in various applications, and challenges you may face in the field. With the right preparation, you can confidently approach your flow cytometry interview and be well prepared to answer any questions that come up.
The challenges of flow cytometry: an interview with David Lanham
If you have a degree in biosciences or biotechnology and technical skills, and you’re looking for an exciting career option in the research sector, go to www. wisdomjobs. com. In call counting, cell sorting, biomarker detection, and protein engineering, flow cytometry uses a laser or impedance-based biophysical technique that suspends cells in a stream of fluid and subjects them to an electronic detection device. In order to determine whether a high white cell count is the result of blood cancer, it is used to analyze blood or bone marrow cells. It can also detect residual levels of disease after treatment. Check out the flow cytometry job interview questions and answers provided to chart your future as a research associate, teacher, operator of flow cytometry, cell analyst, and many other professions.
Flow cytometry is a very flexible method that can be applied to a wide range of applications. It can be used to measure a variety of parameters from a single cell and is very sensitive. It is also a very quick method that can analyze a lot of cells in a short period of time.
The width of the light beam used to excite the cells in a sample during flow cytometry is measured as the aperture size. Smaller apertures produce narrower beams of light, which can be more focused but may also mean that the cells receive less illumination. A wider beam of light is produced by a larger aperture, which can illuminate more cells but may also cause the light to be less focused.
When a fluorochrome (a molecule that absorbs light and then emits it at a different wavelength) is present in a sample but not specifically bound to anything, autofluorescence occurs. In flow cytometry, autofluorescence can result in false positives, or events that appear to be positive for a particular marker but are simply background noise. This can cause issues with data analysis. It is crucial to carefully account for autofluorescence when designing experiments and to use suitable controls when data analysis in order to prevent this.
Because it can reveal a lot of information about the cells in the sample, flow cytometry is an effective tool for analyzing blood samples. These details may relate to the cells’ size, shape, surface markers, and internal composition. Understanding how the blood cells function and diagnosing diseases can both benefit greatly from this knowledge.
The resolution of the data collected by flow cytometry may be enhanced by using multiple lasers. Different colors of light can be used to distinguish between various cell types or subtypes by utilizing a number of lasers. This may help to provide more thorough information regarding the cells being studied.
During your cell analysis, FMO controls, also known as fluorescence minus one controls, are used to position gates. They enable you to gauge how much spread you are experiencing in the negative population in relation to all of the other fluorochromes and post compensation because they contain all of the antibodies in your panel except for one. They are not taken into account when designing the panel, but understanding how the signal spread matrix (SSM) relates to the FMO controls is an important factor.
Check the experiment’s technical aspects first. Are the comparison beads appropriate for the species from which the antibody was isolated? (Not all comparison beads are universal.)
Isotype controls and FMO controls are two different kinds of controls that contribute differently to the experiment. You can determine the spread between channels after compensation using FMO controls. By displaying how much spread there is on the negative population in relation to all the other colors in your experiment, they enable you to precisely decide where to gate for positive populations of cells.
Check the voltages on that particular channel. They might not be ideal if they were predetermined by an automated system. To find a more suitable voltage setting, try titrating the voltages (voltration). Voltages should be raised by 50 in 50-steps starting at 200, and the appearance of the positive peak should be observed. This is your ideal voltage when the distance between positive and negative stops widening. This is how the ideal voltage should be set, perhaps using CD4-stained leukocytes. Try a titration of antibody on the comp beads if this doesn’t resolve the issue. The amount of antibody used to stain beads may differ from the amount used to stain your cells, depending on whether they will produce the right signal for accurate compensation.
There are a few choices here, and they might change depending on the use and whether you intend to fix the cells. Reagents like 7AAD or Draq7 would be useful if you were running unfixed samples. These dyes don’t emit near the fluorescent proteins you’re referring to, but rather in the far-red region of the spectrum. There are amine-reactive fixable viability dyes that emit in the far red that can be used if you want to fix your cells. Examples of these include Zombie-NIR (Biolegend) and Proteintech’s Phantom Dye Red 780 Viability Dye. These are sensitive because the autofluorescence of the living cells provides little background. They can also be applied to live cells, such as when performing cell sorting.
Reflects the scale of the cell. These distinct-sized cells pass through the flow cytometer and over the laser, where the laser collects forward scatter based entirely on how much light is scattered as these cells cross the beam. *Smallest cells = lowest quantity of forward scatter. *Larger cells = maximum ahead scatter.
There is a lot of go-reactivity. Cells might have severa amounts of antigens on them. What is expressed at the green cell can also be expressed at the blue cell, which means that when Ab binds to the green cell in opposition to Ag, it will also bind to the blue cell as well. Cells pass through and have varying amounts of different Abs attached to them.
FAQ
What is the principle of flow cytometry?
A technique called flow cytometry (FCM) enables quick analysis of a large number of statistically significant cells at the single cell level. The main idea behind this method is based on the light scattering and fluorescence emission that take place when a laser beam strikes cells moving in a focused fluid stream.
What are the three major components of a flow cytometer?
Fluidics, optics, and electronics make up the three main parts of a flow cytometer (Figure 1). The flow cytometer’s fluidics system is in charge of moving the sample from the sample tube to the flow cell.
What are the types of flow cytometry?
Fluidics, optics, and electronics are the three main systems that make up a flow cytometer.
What are three possible clinical uses for flow cytometry?
- Immunophenotyping. The most common application performed on the cytometer is immunophenotyping.
- Cell Sorting. …
- Cell Cycle Analysis. …
- Apoptosis. …
- Cell Proliferation Assays. …
- Intracellular Calcium Flux. …
- For More Information.
