Sorting on the MACSQuant Tyto takes place within the completely closed MACSQuant Tyto Cartridge. The cartridge consists of three chambers, input chamber, positive collection chamber, and negative collection chamber.Prior to sorting, fluorescently labeled cells are loaded into the input chamber which contains an adjustable propeller that keeps cells in suspension during the sort. Driven by low air pressure (<3 psi, i.e., <20.7 kPa) applied to the input chamber through a pressure inlet, cells flow from the input chamber, via a microchannel, into the microchip containing the sorting valve. Within the microchip, just before the valve, cells are interrogated by three lasers. Fluorescence and scatter light signatures caused by the laser interrogation are used to distinguish negative cells from target cells. Negative cells are, by default, directed towards the negative collection chamber. To redirect a target cell into the positive collection chamber, a magnetic field is applied to the microchip, which triggers the sorting valve to open and change the flow from the microchip towards the positive collection chamber.
When the MACSQuant Tyto Cartridge is placed in the MACSQuant Tyto Instrument, a solenoid is located adjacent to the microchip. When the solenoid is charged, it attracts the microvalve, which contains a nickel/iron core, thereby changing the direction of the flow within the microchip. By default, cells flow towards the negative collection chamber. When the microvalve moves towards the solenoid, cells are directed into the positive collection chamber. When the charge is removed, a silicon spring returns the microvalve into its original position.Accurate valve opening is crucial to achieve an effective separation of positive and negative cells. To this end, the instrument precisely predicts the arrival of a target cell at the valve.
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.
In an ideal world, cells would be sorted at high speed, purity, and yield at the same time (see figure below, A). In reality, however, the sorting performance is always a compromise between the three parameters. Assuming a constant speed, sorting for highest purity is usually at the expense of yield (B), and, vice versa, sorting for high yield results in lower purity (C). Similarly, if choosing a high sorting speed, purity and yield will be compromised (D). This principle applies to all existing sorting instruments available in the market. On the MACSQuant Tyto either yield, purity, or balanced mode can be selected as a sort algorithm. In yield mode, the sorting algorithm specifically selects all events that are determined as positive events. In purity mode, emphasis is on selecting single, isolated positive events to increase the purity of the positive sort population. Balanced mode reaches a balance between purity and yield. Depending on the application and setting (purity, yield, or balanced mode), purities of 99% and yields >70% can be achieved using the MACSQuant Tyto.Final purity and yield always depend on additional factors such as the cell type being sorted, cell density in the input sample, or the concentration of target cells in the input sample.
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.
The MACSQuant Tyto Cartridge enables a reliable and robust cell sorting 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.
For an overview of different applications for the MACSQuant Tyto, see the applications page on our website, providing data showing sorting of: