Sample Preparation

Tube Requirement for Flow Cytometers:
The Beckman Coulter Cytomics FC 500 Flow Cytometer loads 5.0 ml (12x75mm) Polystyrene or Polypropylene tubes. BD LSR II Flow Cytometer System can only load 5.0 ml (12x75mm) Polystryene tubes. BD InFlux Cell Sorter must use sterile 5.0 ml (12x75mm) Polypropylene tubes. Samples should avoid clumps or aggregates. At least 0.5 ml of cell suspension at a concentration of 0.5 ~ 1 million cells/ml is recommended.

Sample Preparation for Cell Sorting:
Samples should be prepared in a single-cell suspension at a concentration of 4 ~25 million cell/ml. Preservative such as sodium azide should NOT be used. Cells must be filtered through 35~100 um filters just prior to sorting in order to remove any clumps or aggregates. Order information for cell strainers is shown above.

The cells for sorting can be prepared as for flow cytometry analysis, and suspended in PBS or serum-free culture media (no phenol red added) supplemented with a small amount of BSA or FBS (0.5% or 2%) and 2mM EDTA to keep cells viable and non-aggregated.

The facility cannot accept potentially pathogenic samples for cell sorting. If you have questions regarding viable human/primate samples or lentivirus-treated samples, please consult with the Core Manager. Samples labeled with radioactive material are not accepted under any circumstance.

What to bring:
Cells must be kept on ice and 5 ml polypropylene round-bottom tubes 12x75mm style (Falcon #352063 from BD Biosciences). Negative control (unstained cells, 0.5 million/0.5 ml). Single stained cells of each fluorochrome to set up compensation (if multiple staining applied, 0.5 million cells/0.5 ml). Cells to be sorted (4-25 million cells/ml). Additional 5 ml polypropylene round bottom tubes and cell strainer/filters. Cell suspension medium/buffer. Several 5ml round bottom tubes filled with 1ml of culture medium or 50 ml tubes filled with 10 ml of culture medium to collect sorted cells.
A print out of the data obtained on the flow cytometry analyzer which shows your population(s) of interest is preferred.

Sorting Rates:
Suggested sorting rate for cells that are large, fragile or easily aggregate is < 6000/second using 100 micron nozzle. Smaller cells can be sorted at 12,000 ~15,000/second, using 70 micron nozzle.