Flow cytometry and cell sorting are similar techniques with markedly different goals. Both are used to characterize individual cells within a heterogeneous population, but while flow cytometry represents the end of the line for the cells undergoing analysis, cell sorting often provides cells with a new beginning. Here, we look into best practices for ensuring the success of these two established techniques and discuss some modern advances that underpin a growing number of flow cytometry and cell sorting applications.
Different techniques serving different purposes
When considering flow cytometry or cell sorting, it’s important to remember that these techniques are used for different reasons. Flow cytometry serves purely as an analytical tool, meaning that once a given experimental question has been addressed, the cells are discarded. In contrast, the aim of cell sorting is to enrich specific target cells to high purities, allowing for their further use in downstream analyses or applications.
“Flow cytometry employs fluorescently labeled antibodies and the measurement of light scatter properties to examine many different cellular processes and characteristics,” reports Dr. Marco Iodice, scientist at Abcam. “It is widely used for immunophenotyping, whereby the detection of cellular markers provides insights into the expression profiles of cellular subsets. Flow cytometry also allows researchers to monitor changes in cell proliferation and the cell cycle, which has value in drug screening for tumor therapies, drug toxicity, and immune cell proliferation.” Other applications of flow cytometry include analysis of cellular signaling pathways and detection of apoptosis.
Samson Rogers, co-founder and CEO of TTP spinout Cellular Highways, notes that sorted cells are used for genetic analysis, for assays requiring live cells, and for subculture or cell-line development. He also points out that cell sorting has vast potential for cellular therapies but, to date, this has been inhibited by several major limitations of the technology. “Conventional cell sorting relies on electrostatic steering of droplets flying through the air,” he explains. “It is therefore non-sterile, biohazardous due to the aerosol risk, limited on speed, damaging to many cell types, and requires an expert operator.”
Felix Eppler, Ph.D., global product manager for cell sorting at Miltenyi Biotec, agrees, adding that cell damage results from high pressures, strong decompression forces, the application of electrical charge, and high shear forces. “It’s no secret that the combination of these factors can lead to poor cellular viabilities and functionalities after the sorting process,” he says. “Moreover, because the internal fluidics of conventional cell sorting systems are shared between samples, there is a high risk of contamination or carry over that can compromise sample quality.”
Best practices for flow cytometry and cell sorting success
With both flow cytometry and cell sorting requiring single cell suspensions, several shared best practices are essential. “Although cells will not be recovered at the end of a flow cytometry experiment, cell health and viability must still be maintained,” says Iodice. “Cell death can switch on numerous cellular pathways with the capacity to alter cellular behaviors and impact the target of interest, and it also releases a plethora of biochemicals that can influence surrounding cells.” Steps for ensuring cell viability include lowering the temperature to decrease metabolic activity; adding a protein source to buffers to stabilize cells; and reducing any physical or shear stresses resulting from pipetting or centrifugation. Additional measures involve passing cells through a nylon mesh filter and including EDTA in buffers to eliminate clumps; optimizing cell numbers for the experimental system, with 1-10 x106 cells/mL being a good starting point; and using viability dyes to distinguish live and dead cells. Time may also be a factor if cells are required to undergo extended treatments or incubations.
Modern technologies opening up new possibilities
Many innovative developments have helped to improve the scope and reliability of flow cytometry and cell sorting in recent years. Fluorescent labels within the UV and IR range, along with novel tandem dyes, have increased panel sizes for greater multiplexing capabilities, while the majority of antibody manufacturers have invested considerable effort to improve the validation (and consequently the performance) of their reagents. Many of Abcam’s conjugated RabMAb® antibodies have been validated for specificity against knockout cell lines, including application-specific validation using flow cytometry, whereas Miltenyi’s REAlease® Releasable Antibody Technology provides easy removal of antibodies after cell labeling. “Depending on the downstream application, it can be important to remove bound antibodies from your cells,” says Eppler. “For example, this might be required if cells are to be activated, if certain epitopes need to be made accessible again, or if cells are to be transplanted into patients or animal models.”
One area seeing the greatest disruption is that of cell sorting instrumentation, with manufacturers promising faster, gentler, scalable, sterile sorting to support a growing range of applications. Olivier Déry, director of marketing at NanoCellect Biomedical, explains the benefits of a benchtop solution. “Flow cytometers and cell isolation products are typically very bulky and require a lot of specialized equipment, meaning many users have to book time at a core facility to carry out their experiments,” he says. “Our WOLF® Cell Sorter has a footprint of less than 2 cubic feet, allowing it to be placed anywhere in the lab—even in a hood—and it is readily paired with our N1 Single Cell Dispenser to transfer sorted cells into 96 and 384 multiwell plates. Not only does the small footprint provide researchers with greater flexibility, but because sorting takes place within a disposable microfluidic cartridge, sample-to-sample contamination and biohazard aerosols are eliminated.”
Cellular Highways’ cell sorting technology, known as vortex actuated cell sorting (VACS), also overcomes the problems of sample contamination and aerosol generation. “VACS was designed to provide faster aseptic cell sorting compared to conventional instrumentation,” says Rogers. “This is achieved through parallelization, whereby a chip containing multiple sorters is used to scale cell sorting to incredibly high throughput. We demonstrated a 16x parallel chip at CYTO2019, capable of providing an approximate 10-fold speed improvement on leading available sorters without compromising cell health. This has received a lot of interest, especially from those with a requirement for cell populations with high viability, purity, and yield for use within cell therapies and other fields.”
Image:Neutrophil granulocytes sorted from non-lyzed whole blood using the MACSQuant Tyto Cell Sorter have high purity and functionality. Image provided by Miltenyi Biotec.
Eppler notes that one of the advantages of Miltenyi’s MACSQuant® Tyto® Cell Sorter is that it can be used to sort even very delicate cells. “Cells such as neutrophil granulocytes are extremely sensitive to environmental factors like mechanical stress and fluctuating temperature,” he says. “Therefore, gentle sorting is critical for accurate analysis of their function. By using a microvalve to sort cells inside a closed cartridge system, the MACSQuant Tyto provides a low pressure, sterile, and temperature-controlled sorting environment that means cells remain highly functional. Moreover, because the plug-and-play cartridge system is so intuitive, even those with little experience in flow cytometry are able to use multiparametric cell sorting for their research.”