Sample preparation for sorting

1. Available nozzles on our BD FACSAria sorter and recommendations for samples:

The lower the pressure the better for the survival of your cells J

2. Our sorter is equipped in following tube/plate holders:

4x 1.5 mL/2 mL micro tubes

4x 5mL FACS tubes

2x 5mL FACS tubes

2x 15 mL Falcon tubes

Multiwell plates (6/12/24/96/384 well)

Microscope slide

3. You should bring samples for sorting in 5 mL FACS tubes in minimum sample volume exceeding 200 µL.

4. Depending on the used nozzle, the good cell concentrations to start with are:

Please always bring buffer to be able to dilute the cell’s suspension further if required.

5. Always sort into vessels containing collection medium!!! Prepare collection tubes/plates with sufficient collection medium with regard to the expected yield.

Collection medium depends on your downstream application.

Harmful volatile substances are not allowed as collection medium in our facility.

6. To estimate background signal consider to use unstained / mock transfected / single transfected cells. Additionally, for multicolor experiments, very useful and helpful are FMO and compensation controls (single stained samples) depending on your panel requirements. In case of doubt, please do not hesitate to contact us.

7. Remember: Always filter samples JUST before sorting!!! For this purpose use FACS tubes with blue caps.

If your cells have a tendency to form aggregates/clumps, you can always use (if possible) one of the following modifications:

⇒ use calcium/magnesium free buffer

⇒ add EDTA (2 - 5 mM)

⇒ add 25 µg/ml DNAse I + 5 mM MgCl₂ (no EDTA then), if the cell preparation induces increased cell lysis

⇒ add 1% Accutase in sorting buffer