Frequently Asked Questions

How do you prepare samples for flow cytometry?
Count the cells and check cell viability. The recommended cell concentration for analysis (sorting needs more cells) is 1-2×106 cells/sample. Samples must be filtered and provided in a 12 x 75mm tube. The aggregates can be removed by filtration using commercially available filters including the 35 micron FALCON strainer cap/test tube (35-2235) or the 40 micron FALCON cell strainer (32-2340).

What kind of controls should I have?
For each cell type examined, bring negative controls, compensation controls, gating controls and experimental controls. Please refer to the form below. For gating controls (FMO, Fluorescence Minus One), please refer the resource: About gating control FMO .

1	Negative Controls	Unstained cells (no fluorochrome/dye added).
		No-specific staining controls (e.g., cells were stained with only 2ndAb, no primary Ab; mock transfected cells).
2	Compensation Controls	Single stained cells (stained with only one fluorochrome/dye and must have both negative and positive cell population) one for each fluorochrome/dye used in the experiment.
3	Gating controls (FMO)	Cells stained with all fluorochromes/dyes except one, leave out one dye at a time.
4	Experimental Controls	According to your experiments, fully stained healthy or untreated cells.

How many cells I should prepare for sorting?
You have to calculate backwards: how many sorted cells you need; what percentage of the cells you are interested in from the total population; the viability of the cells before and after sorting. For example: you need 1×106 sorted cells, the approximate percentage of interested cells is 50 percent of the total cell population, 5 percent of the cells were dead before sorting, so the least number of cells you have to prepare are: 1 × 106 /0.5/(1 - 0.05 ) = 2.1 × 106 (it assumes that all the sorted cells are alive.) If your cells are fragile, 50 percent of them may die after sorting, then you have to bring at least 4.2×106 (2.1×106 /0.5) cells. 0.5 - 1.0 ug/mL viability dye can be added to the sample in order to sort only live cells.

How do you determine the purity of the sorted cells?
The purity can be determined by the post-sort analysis of the sorted cells.

What is the maximum purity that can be obtained after sorting?
Ideally, the maximum purity is 99-100 percent and it is easily achieved when using beads. Usually more than 95 percent purity can be expected if the interested population is well separated from the unwanted cells.

Is it possible to re-culture the sorted cells?
Yes, as long as you request a sterile sort. Although sorting on the flow cytometer is kind of under open air, we can autoclave the sheath solution and use 70 percent ethanol to clean the sort area so the chance of contamination is less. Be sure to put the sorted cells in the medium with antibiotics, which also prevents contamination.

What is the optimal cell concentration for sorting?
It can range from 3 x 106 - 15 x 106 cells/mL. We recommend 8 - 10 × 106 cells/mL, which will allow to sort at low flow rate and improve purity and yield.

What kind of medium should I use for sorting?
We use sterile PBS containing 2 percent FBS and 0.1 percent sodium azide.

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