Seeing is Believing – How Cell Images Enable More Accuracy in Flow Cytometry Data?

Common Challenges in Flow Cytometry

Flow cytometry is one of the most powerful tools available today for multiparametric single cell analysis in various disciplines. Particularly, immunological research, helps to understand complex mechanisms through the study of cells of interest among a mix of cell populations. But, like many other good things in life, flow cytometry is not without its drawbacks which affect the quality of the data obtained. For instance, the presence of aggregates/doublets in the sample could result in erroneous data and dead cells haunt researchers by nonspecifically binding to reagents and giving false positives. Data acquired from traditional flow cytometry does not provide any morphological data or visual images, particularly important in immunology to study cell interactions, and maintaining quality while processing live cell cultures.

Which Solutions are Available?

Thankfully, scientific advancements, particularly in immunology, provide some great solutions today, like viability dyes that differentiate the live cells from the dead or gating strategies that keep doublets out of the analysis. However, these solutions lead to some newer challenges. Viability dye is an additional reagent which wouldn’t be otherwise needed, so it adds an extra step and cost. Who needs extra cost in this competitive market? Also, this solution blocks a channel for detection of viability dye’s fluorescence. It takes away one parameter for analysis if using a full panel. So much for having a multi-channel flow cytometry.

In short, these solutions do offer advantages but not without some compromises. Another ideal option would be to get a clean sample of just viable single cells. But let’s be real, it’s not always possible to get an ideal sample, or even enough sample, as it often comes from human patients. After all, it’s not feasible for analysts to go back to already suffering patients and ask for more of their tissue.

Is There a Better Way Out?

Just imagine if an instrument existed that could take pictures of cells during flow cytometry and revealed if they are doublets or dead cells. Well, good news, Thermo Fisher Scientific has made that imagination a reality with their Invitrogen^TM Attune^TM CytPix^TM Flow Cytometer its high-speed brightfield camera coupled with Attune Cytometric software can take pictures of each acquired event and help distinguish Single cells from doublets, clumps dead cells or debris. Bonus point, image capture happens when the sample flows through the flow cell. This technology removes unwanted cells and acquires highly accurate data, specifically for the target cell populations.

One might wonder, how many images can one instrument take? The answer is a lot! Its high-speed camera can take up to 6,000 images per second, increasing the scope of analysis to a great extent. Acoutic focusing further enhances image quality. Visual images provided by Attune CytPix model also unlocks the potential for improving cell culture quality check (QC). For example, in one study cell culture QC scientists observed loss of viability in terms of reduced cell counts and survival despite appearing confluent. Using previously captured images by Attune CytPix model they could find the root cause as a microbial contamination. It wouldn’t have been possible without these images. Another advantage is the ability to observe cell-to-cell interactions. When CAR-T immunotherapy cells were co-incubated with Ramos (lymphoma) cells and processed using Attune CytPix model, some images showed CAR-T cells visibly targeting Ramos cells.

This solution could be a game changer. It eliminates the need for any additional reagent such as viability dyes, which saves money and doesn’t block any channels. This allows maximum parameters to be used for the actual research study, while also cleaning up the mess from doublets and dead cells. In addition, it can unlock opportunities for improving QC processes, studying cell-to-cell interactions and much more.

Could this be a dream instrument for improvement of data accuracy and simplified immunology research?

Learn more about the product in this blog: Invitrogen Attune CytPix Flow Cytometer

Resources and Solutions for Flow Cytometry

• Flow Cytometry Panel Builder – this online tool helps you design an effective reagent panel for your specific application on your conventional or spectral flow cytometer
• Flow Cytometry Protocols Handbook – protocols that fit your needs in flow cytometry ranging from sample preparation to numerous cell stimulation conditions, staining, immunophenotyping and data analysis strategies
• Immunology at Work Resource Center – practical guidance and protocols to help you get started quickly and confidently

References

1. How experts are breaking down barriers in flow cytometry (selectscience.net)

Additional Blogs

• Clog Resistance of non-Pressure Based Flow Cytometers

• Is it a T-Cell or B-Cell? Antibodies for Immunophenotyping