FACS Calibur

Flow Cytometry with the FACSCalibur

Take to the FACS

- The cells to be sorted, appropriately diluted (more on that later)
- Empty FACS tubes (Sarstedt cat. no. 55.1579)
- A bottle of PBS (you need 1 mL per sample)
- Pipettes (1 mL and 10 µl)
- Pipette tips (a few 1-mL tips and a 10-µl tip for every sample)
- USB memory stick

Preparation of samples

Overnight cultures: Dilute 100-fold in PBS (100 µl sample are more than enough). To be on the safe side, do a 10- and a 100-fold dilution and take both with you.

Cultures grown on a plate: Use PBS to wash the cells of the plate (tilt the plate and repeatedly pipette the PBS to wash all colonies (or a portion of them) off the plate. Depending on the finally concentration, you will have to dilute the cell suspension 10- or 100- fold.

Start up

Instructions on how to start up the machine and the computer can be found next to the FACS.

1. File > Open. Open a Page Layout that you have made previously (E. coli setup).
2. Acquire > Connect to Cytometer (command-B). Also press Command-1, Command-2, Command-3, and Command-4 (these windows are not strictly necessary, but allow you to monitor various parameters).
3. Acquire > Acqusition and Storage. Indicate the number of events you want to to acquire (I acquire 20'000 events).
4. Acquire > Parameter description. Select the correct folder where your samples will be stored and set the naming and numbering of your samples.
5. Cytometer > Instrument Settings > Open. Open a file from a previous session and click Set after opening the correct file. Click Done.
6. Acquire > Counters.
7. Wait for the “Ready” or “Standby” signal on the status view.
8. Check the name of the sample, uncheck Setup, and acquire on Lo. If there are problems, it could be the pressure. Change to Med speed if needed. Cell flow should not exceed 3000 events per second.
9. When finished collecting data, transfer your files to a USB stick and erase any old files. Please do not store your data on this computer.