The MACSQuant Tyto Sorter

To anticipate when a target cell will be arriving at the valve, the time of flight (TOF), and thus the speed, is determined. To define the TOF, the time it takes for a cell to travel between two adjacent lasers is measured on two photomultiplier tube (PMT) channels, the so-called trigger channel and cell speed channel. The trigger channel is used to set the threshold that instructs the instrument as to what should be considered a positive event, i.e., a target cell.

Due to differences in the flow speed within the microchannel of the MACSQuant Tyto Cartridge chip, trigger and cell speed channels are preferably selected based on adjacent lasers closest to the sort valve. This setup enables optimal cell speed determination. Below figure shows a schematic overview of recommended trigger/cell speed laser combinations.

 A short instruction with detailed information on valve timing can be downloaded from the related resources panel to the right[MO1].

• Instrument can be operated easily by lab professionals without intensive training.
• Cartridge identification is performed by a barcode scanner, which automatically detects cartridge type and lot.
• Loading of the cartridge into the instruments occurs in an intuitive “plug-and-play” fashion.
• Flow control, laser alignment, speed detection, and valve timing are fully automated.
• The automated sort setup function for a variety of applications adds to the ease of use. Actual sorting process is completely hands-off, saving time, resources, and thus money.

How do all these features add up in the overall hands-on time? Below table summarizes a side-by-side comparison between the workflows of the MACSQuant Tyto and a conventional droplet sorter.

The technology behind the MACSQuant Tyto is unique in its kind. Below table points out the key features and benefits of the MACSQuant Tyto compared to conventional droplet sorters.

At a glance: Technological features and benefits of the MACSQuant Tyto Sorter compared to coventional droplet sorters

The MACSQuant Tyto Cartridge ensures a high level of safety during sorting.

• All chambers of the MACSQuant Tyto Cartridge contain 0.1 µm hydrophobic filters allowing for sterile ventilation of the sample
• Air used for pressurizing the input chamber to control the sorting flow is pre-filtered.
• All MACSQuant Tyto Cartridges undergo a validated and strictly monitored sterilization process using ethylene oxide, ensuring full sterility. 

The MACSQuant Tyto Cartridge enables a reliable and robust cell sorting process.

• 3D filter is located within the input chamber in front of the microchannel to retain larger particles and aggregates and prevent blockage of the sorting valve.
• In the unlikely event of a clog in the microchannel, an instant, high-vibrating fluid stream helps to dissolve the clog.
• In case of a persistent clog of the microchannel, the MACSQuant Tyto Cartridge, in contrast to conventional droplet sorters, allows for complete recovery of the sample without risk of cross-contamination of the sorted fraction. The remaining sample simply needs to be removed from the input chamber and loaded into a new cartridge to continue the sort process.  
• During the sort, automated laser re-alignment is executed continuously based on marks on the microchip. This ensures optimal sort conditions during the entire process.
• Automated flow control, speed detection, and valve timing allow for a completely operator-free sort process.

During the sort of a particular target cell population on the MACSQuant Tyto, the unsorted non-target cells are collected in the negative sorting chamber, thus available for further sorting steps. This feature serves three purposes.

• Rare target cell populations (<0.05% of original sample) can be sorted to high purity by performing a two-step sort: an initial sorting with emphasis on high yield, followed by an additional sorting aiming at high purity.
• To increase the yield after an initial sorting round aiming at purity, the negative population can be processed in the same way to obtain even more target cells out of this population. This method can be helpful for the isolation of rare cell populations such as tumor-infiltrating lymphocytes (TILs) or when using precious FFPE material. 
• The MACSQuant Tyto allows for sorting of multiple populations out of a single sample. Since the negative fraction is preserved after a sort, this fraction can be subsequently sorted to obtain a functionally different population.