High-Throughput Plate Sorting

High-speed electrostatic droplet sorting is the standard technique for cell purification across a wide breadth of research areas in the biological sciences and beyond. Purified cells are utilized for an array of downstream experiments, including functional assays, DNA analysis, gene expression analysis, and cell line development. Some of these key applications, especially gene expression analysis through RNA sequencing and clonal cell line development, require direct deposition of single cells into multiwell plates. Microtiter plate sorting is a powerful capability that takes advantage of the inherent single-cell resolution of flow cytometry and droplet-based cell sorting, but it presents unique challenges. Specifically, cells must be accurately and precisely targeted into each well to ensure that cells are deposited directly into potentially small volumes of capture fluid.

Plate sorting is accomplished through movement of a stage that holds the plate, positioning it directly under a sort stream that will deliver a cell into each well. Because of the requirement for precise targeting, the plate deposition mechanism requires careful calibration and alignment. Alignment is commonly verified by sorting a small number of droplets onto the cover of the plate and ensuring that the drops are positioned in the center of the well as viewed from the top of the plate. However, this method is imperfect, especially for deposition into PCR plates.

An alternative methods have been developed to verify plate alignment. One such method, developed by Rodrigues and Monard [1], utilizes horseradish peroxidase (HRP) to catalyze a colorimetric reaction, which indicates whether a droplet has been deposited into a well. In this method, a plate is filled with a small volume of buffer containing the colorless substrate 3,3,5,5-tetramethylbenzidine (TMB), and beads suspended in a solution containing HRP are sorted into each well. TMB turns blue after reacting with HRP, so any color change after sorting indicates successful droplet deposition.

The Invitrogen™ Bigfoot Spectral Cell Sorter is a cuttingedge, high-parameter instrument that features a multitude of cell-sorting advancements. Innovations in plate deposition ensure unprecedented accuracy, recovery, and speed down to single-cell sorting. In this paper a variety of tests using the horseradish peroxidase (HRP) method were performed on the Bigfoot Spectral Cell Sorter to test deposition accuracy and precision, robustness, and speed of the hardware.