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Designing Increasingly Complex Flow Cytometry Panels for High-Throughput Immunophenotyping

Offering simultaneous detection of an expanding array of cell markers, flow cytometry has become an indispensable high-throughput tool, used for the detection and quantification of diverse immune cell populations within heterogeneous samples. With a broad range of research and clinical applications, this analytical technique plays a pivotal role in understanding the immune system’s role in disease progression, monitoring patient responses to treatment, and assessing the safety and efficacy of novel therapies and vaccines. Careful panel design is crucial to ensuring data accuracy, particularly when performing complex multiparameter assays.1,2

To ensure panel building success, a selection of best practice guidelines surrounding core aspects of immunophenotyping can be followed to assist with experiment design.

General Sample Preparation Tips

To ensure the success of your flow cytometry experiments, it’s essential to prioritise meticulous sample preparation. Investing extra time in this phase can yield significantly improved results, saving you from the frustration of failed experiments and the need for multiple repetitions to achieve publication-quality data.3

Select Appropriate Tubes: Choosing the right tubes for your flow cytometry experiments is crucial. Some cytometer models allow flexibility in tube selection, like Bio-Rad’s ZE5 Cell Analyzer, but others have restrictions. It’s important to use tubes that are compatible with your cytometer model, considering factors such as round or flat bottoms and the type of plastics used. Additionally, be mindful of cell adhesion to different plastic materials. For instance, certain cell types, like monocytes, may adhere more strongly to polystyrene than polypropylene. Selecting the appropriate plastic can help minimise cell loss and the need for harsh detachment methods.

Handle Samples Gently: Maintaining the optimal condition of your cells is paramount. Avoid subjecting them to unnecessarily harsh conditions that can lead to cell death or generate artifacts, as this can significantly impact your results. When centrifuging samples, be cautious not to spin them at higher speeds than necessary and avoid leaving samples in the centrifuge for extended periods, as prolonged pelleting can be detrimental. It’s advisable to handle samples gently to prevent the formation of bubbles, which can harm cells. When aspirating media, ensure that you leave some supernatant behind to prevent cells from drying out in a pellet.

Prevent Clumping: Flow cytometry relies on analysing individual, single cells. Clumps of cells can obstruct the flow cytometer, causing clogs and inconvenience to other users. To reduce the risk of clumping, consider keeping wash and media buffers at a temperature of 4°C, particularly if your experiment permits this.

Dissociate Tissues Thoughtfully: Achieving single, intact cells suitable for flow cytometry is essential. When dissociating tissues, take care to minimise cell damage. Consider methods such as tissue mincing, mesh filtering, or enzymatic digestion based on the specific requirements of your cell type. If you are working with rare cell types, opt for gentler methods to preserve them. For example, neutral protease (dispase) is a milder treatment than trypsin and is commonly used for isolating iPSCs. Some cell types, like F4/80+ macrophages and follicular dendritic cells, may require enzymatic treatment for release, and the specific enzymes needed should be determined based on established protocols.