MACSQuant Tyto

Cartridge-based cell sorter

Overview

The MACSQuant Tyto is built around the idea of keeping the sample separated from contamination sources – or, inversely, keeping the sample from contaminating anything else. Cell sorting on a MACSQuant Tyto occurs entirely in a closed cartridge that is divided in to three chambers: the input, positive collection, and negative collection.

In the comfort of a sterile tissue culture flow hood, the user loads the sample in to the sample chamber. Once the cartridge is sealed, nothing goes in or out of it until the sort is complete. The user can then bring the cartridge to the instrument and install it in the sample block.

To sort the cells, low air pressure (< 3 psi) is applied to the sample, which is then pushed through a fluidic chip. The cells are interrogated by the lasers, the signals are processed, and cells within the target population are diverted into the positive chamber by a high frequency valve. The unsorted flow through continues towards the negative chamber.

Main Applications

Sterility

Ideal solution when sorted cells need to go back in culture

Biosaftey

Provide complete bio-hazard protection to the operator

Fragile Cells

Fragile samples will fare better sorting at low 3psi pressure (for comparison, low pressure on the Aria is ~30psi)

Resources

The MACSQuant Tyto is equipped with three 100mW lasers and 8 fluorophore detectors, plus the SSC and backscatter detectors:

488nm: 525/50, 585/40, 693/38 , 750LP
405nm: 450/50, 510/50
635nm: 693/38, 750LP

For best results, it is ideal to assign to highly expressed markers with very distinct on/off pattern of expression. Signal emission and light collection is impaired by the cartridge. B4 and R2 (PECy7 and APCCy7) are not suitable for dim marker and should be used for bright, primary markers. Bright fluorophores on better channels such as V1, V2, B1 and B2 (BV421, BV510, BB515 and PE respectively) are preferred to resolve secondary and tertiary markers. Note that FSC and SSC will not be appropriate for defining populations of cells, they must be defined/gated by a fluorescent marker.

Sort purity is a function of cell concentration on the Tyto

The flow rate on the Tyto is fixed to 4mL per hour, so event rate is mainly controlled by the cell concentration of the sample. The highest recommended sort rate is 5000 cells per second. so Nicely enough, the sample preparation can be optimized beforehand by calculations based on Poisson distribution. The frequency of the target population of cells and the desired sort purity will dictate the most favorable cell concentration or inversely, the cell concentration and population frequency will predict the sort purity.

Download the Tyto Cell Concentration Calculator to select the optimal cell concentration for your assay.

Tyto Concentration Calculator

Once the sort is completed, a purity check should be performed to assess the result. Running the collected cells in a new cartridge is not a viable solution. We recommend sampling the collected cells on a benchtop instrument that will provide a true cell count, such as the Attune NxT or the Penteon.

For best result, you should run:

a fraction of the sample
a fraction of the collected cells

The Attune NxT and the Penteon will provide a value for your cell concentration and you will be able to know how many cells have been collected. Purity of the sort will be assessed but the display of your cells on the graphs: a pure sort should give tight clusters of cells with few (if any) outliers.

Tutorial

Unassisted cell sorting

$30/hour

Charged based on time used
The instrument will run 4mL/hr – mind the cell concentration for best results
Staff time will be charged for initial template settings
Once a template is designed, the instrument can be used without involvement from the staff

Cartridges

$100/each

Cartridges are available from the CAT facility at a discounted price
Cartridges should be single use only

Cytometry and Antibody Technology