Sort Calculator by Percent

Rmax is an estimate of sort recovery influenced by several factors including;

1) How evenly illuminated the particles are through the laser beam,

2) How discrete the populations appear in the scatter plots,

3) How well the electronics are dealing with the identification and sorting of a target population in a sample,

4) How well prepared the sample is, ie., the sample is mono-dispersed.

The Centre Stream Catch (CSC) is a sample of the waste stream during a sort. Any target particle not sorted will end up in the waste stream.

The following simplified expression for Rmax% can be use if the original %target is less than 5%:

Where O_t and O_nt are target and non-target in the original sample whereas C_t/C_nt are target and non-target in the Centre Stream Catch.

Data Collection

Set up a sort Target(s) only one-drop sort mode. Sort beads made up of blank beads and target fluorescent beads (less than 5%). Sonicate/Vortex so the beads should be mostly singlets. Run the beads at an event rate about 1/10 to 1/20 the drop generation rate.

Sort the target and at the same time collect the CSC. Fill a 5 mL tube with the CSC and centrifuge. Decant the supernatant to leave about 300µl, analyse, and record the %target. Enter the original %target and the %CSC target in the calculator. The resultant Rmax evaluates the instrument and/or experiment performance.

Results

The higher the Rmax the more Poisson like the particles behaviour. Run the beads first to establish how well the instrument is performing by assuming that they are behaving in an idealised Poisson way.

If the Rmax is low (less than 90%) then investigate the instrument for instabilities, such as fluidic instabilities or electronic issues with the sort or an incorrect drop delay etc.

If the Rmax is 90% or higher, then run the experimental sample.

Calculating Rmax on the experiment evaluates whether the particles are behaving in a Poisson way, ie., that the sample is a single particle suspension.

If the Rmax is low compared to the bead Rmax look into the sample’s preparation to reduce particle clumping

Rmax can also be used to set the optimal drop delay. Set the initial drop delay and collect the CSC. Alter the delay by small steps (fractions of a drop) and collect the CSC. The delay with the highest Rmax value is optimal drop delay