Evaluating Cell Sorting Parameters

Cell sorting is a powerful tool that allows a user to isolate a single cell from a culture sample. Though this is a powerful technique, errors in the sorting process can oftentimes cause a scientist much grief in both time and money. No one wants to lose their precious cells after weeks of labor, so having a confident sorting scheme is essential for downstream molecular and cellular processes. Using flow cytometry to sort has advantages over other sorting processes, such as higher purity, separation based on level of fluorescence, employing multiple Ab for better separation, improved recovery and live/dead screening, among others.
 
Depositing a single cell in a consistent volume within a well is typically the goal, but how does one efficiently evaluate the changes made during optimization of the sorting process?  Thankfully, researchers have developed an effective way to assess changes made to an instrument sorting procedure.
 
To assess the volume deposition, the method exploits the HRP-TMB reaction typically employed in ELISA analysis. In the presence of H2O2, HRP reduces hydrogen peroxide yielding water and oxygen by oxidizing TMB. Oxidized TMB results in a color change from colorless to blue, the more constituents available to react, the darker the color. If Well A1 in the plate receives 30% more volume, or receives two drops instead of one compared to Well A2, then the solution in well A2 will be darker blue. A plate reader can discern these differences with the requisite accuracy.

The cells are represented by fluorescent particles that can be observed microscopically to confirm single particle deposition. Ideally, the operator wants one bead deposited per well. If the particle is fluorescent, an inverted fluorescent microscope could be used to observe particle deposition.

General Protocol Steps (specifics dependent upon instrument and project):
1.     Suspend particles in buffer containing HRP
2.     Prepare plate containing TMB
3.     Sort cells and evaluate - a single drop will effect a color change
4.     The degree of color change informs the user about volume deposition while the beads represent cells
 
This technique is a cost-effective way an operator can efficiently evaluate sorting without losing valuable and more expensive cells.

Rodrigues, O. R., & Monard, S. (2016). A rapid method to verify single-cell deposition setup for cell sorters. Cytometry Part A, 89(6), 594-600. doi:10.1002/cyto.a.22865
Osborne, G. W. (2011). Recent Advances in Flow Cytometric Cell Sorting. Methods in Cell Biology Recent Advances in Cytometry, Part A - Instrumentation, Methods, 533-556. doi:10.1016/b978-0-12-374912-3.00021-3