FACS - fluorescence activated cell sorting
The ability to select any population, defined by a logical combination of regions, and isolate that population from the sample.
How to sort cells?
The initial processes are the same as for cytometric analysis:
hydrodynamic focusing of the cell or particle mixture to form a central core in the fluid coating;
passing the cells through the laser beam with subsequent analysis of the scattering and fluorescence signals;
using regions and gates to define subpopulations;
Electrostatic sorting: course of events in the sorting chamber?
1) Vibration of the nozzle under the influence of the transducer causes the stream to break down into droplets containing sorted cells / particles;
2) At the interrogation point level (where the laser beam runs), the signal is processed and the decision made: sort to the left, sort to the right, discard;
3) Stream loading: negative charge when the drop goes left; positive when right;
4) Bending of the stream in the electrostatic field created by the deflection plates maintained at high voltage (+/- 5000 V): the charged drops are attracted to the appropriate plates, forming side fluxes;
5) Collecting sorted droplets of fluid containing cells / particles into tubes or onto a plate.
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)
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