BRC Cambridge Immunophenotyping Hub
SOP for operation of the BD-FACSJazz cell sorter
The FACSJazz consists of
1Contained Air Systems BioMat2 class 2 MSC (Microbiological Safety Cabinet)
2Cell Sorter Unit inside the MSC
3 Waste and sheath tanks contained inside the MSC
4External Server and Client Computers
Wet Waste
The waste generated by the Jazz collects in a vacuum sealed stainless steel tank. This waste is emptied down a dedicated sink, some tablets of Virkon are added to the empty tank before reconnection. The waste tank is left unpressurised in the hood after the machine is shut down, and emptied the following day.
Sheath Fluid
The Jazz runs on a PBS based saline buffer, generally BD FACSFlow. The Sheath Tank should be filled below the metal-seam limit
Start-up
The various units of the machine are turned on in the following order
MSC, Server, Cell Sorter, Client Computer.
The front areas of the sorter should be sprayed with 70%Ethanol prior to alignment and operation. The MSC should operate for 30m before running any class 2 biological material. In addition, the 5kW A/C unit on the opposite wall to the Jazz should be turned off during class 2 sorts
The cell sorter unit, sheath and waste tanks should be placed 12" away from the front opening of the MSC
Alignment and Operation
The front area of the sorter unit should be cleared of obstruction prior to launching BD FACS sortware. This software connects the server, client and sorter units
The pre-stream fluidics functions are applied in the order
Rinse, Purge, Pulse
Prior to these steps, the 'crack-pipe' unit under the nozzle should be filled to overflowing with either FACSFlow or 70% Ethanol.
The Rinse function opens the valves between the sheath and waste tanks. As Rinsing progresses the sheath filter should be agitated until any air is purged to waste.
The Purge function similarly removes air from the nozzle volume, Purging sends fluid from the crack-pipe through parallel tubing
The Pulse function operates during Purging and aids the removal of smaller air bubbles. After all air is removed, the Purge is deactivated and the crack-pipe assembly removed allowing the stream to be turned on.
The alignment of the machine proceeds initally by eye.
The gold micrometer controls align the stream to the waste pipe
The silver micrometer controls align the stream to the laser intercept
Fine alignment is achieved by 2 rotating prisms (1 per laser). These prisms are rotated in combination with the gold and silver controls while fluorescent microspheres are run as a sample.
Drop Delay Calibration
Drop Delay is set using accudrop beads or similar red excited beads.
Prior to running the beads the phase of deflection should be optimised using a test pattern. The amplitude of the test deflection should match the desired deflection for subsequent sample sorting. In addition, the alignment of the accudrop red laser beam should be optimised with this test pattern on.
All thebeads are set to be deflected to the left, while the drop delay is varied with a 3drop window. When the delay is found approximately, the stringency of the sort decision is changed to 1 drop purity until the correct drop delay is found
FACSRinse detergent is run for 5 minutes, followed by 5 minutes backflushing prior to the introduction of biological samples.
Blockage
In the event of a blockage or machine malfunction, the stream and plates are turned off. All surfaces of the sample port and sorting chamber are sprayed with 70% Ethanol. The machine is then left for 5m.
The nozzle or other blocked part is then removed and resprayed with ethanol, prior to sonication or syringing
Other Maintenance
When parts of the FACSJazz are to be inspected or replaced, The MSC should be left on throughout the procedure. Any discarded components should be autoclaved. Tools used for maintenance should be sprayed with ethanol.
Simon McCallum1st September 2014