Flow Cytometry Core Service

In March 2021, the facility took delivery of a spectral analyser, the Cytek ® Aurora. This is a 3 laser machine, with a 96 well plate loader, capable of detecting up to 20 fluorochromes simultaneously.

flow@sheffield.ac.uk

• Bookings and charges
• Background
• Cell sorting
• Filters and configurations

Services provided by the facility

Experimental design

Advice regarding controls, dyes, fluorochrome choice, cell number and staining protocols can be provided. 

Training

Users can undertake a 3 session training course and then be signed off to run samples independently on the analysers and the FACSMelody cell sorter. Please contact the facility at flow@sheffield.ac.uk to arrange this. 

For an additional charge, users can request for a member of the flow cytometry facility staff to run samples.

Analysis

Advice can be provided to assist with data analysis, using both the machine’s acquisition-analysis software, FlowJo or FCSExpress analysis software.

Machines available

Analysers

• BDFACSCalibur – 488 and 633nm lasers. 4 detectors. Tube loading only.

• BDLSRII – 355, 405, 488 and 633nm lasers. 13 detectors. Tube loading only.

• Cytek Aurora – 405, 488 and 633nm lasers. 38 detection channels. Tube and plate loading.

Sorters

• BDFACSMelody - 488, 561 and 633nm lasers. 8 detectors. Tube loading only. Sort 2 populations into tubes simulataneously, sort cells into plates (24, 48, 96 and 384)

• BDFACSAriaIIu – 405, 488 and 633nm lasers. 9 detectors. Tube loading only. Sort 4 populations into tubes simulataneously, sort cells into plates (24, 48, 96 and 384)

Miscellaneous

• gentleMACS dissociator – automated tissue dissociator seeperating organs and tissues into sterile single cell suspensions
• Cytometric Bead Array assays – a technique to quantify multiple proteins simultaneously. Please contact the facility for further information.

Background

Flow cytometry is a technique used for measuring and analysing certain characteristics of a single particle or cell. The particles or cells move through a fluid system and pass through a laser where the relative size, granularity / internal complexity and the fluorescence intensity associated with the particle are measured. A Flow Cytometer comprises a fluidics, optics and electronics systems.

Optics

A series of prisms and lenses guides laser light to the cuvette where it intercepts the hydrodynamically focused sample stream. At this point any fluorescence associated with the particle and scatter information of the particle can be obtained.

Light scattered in the forward direction is collected by the Forward Scatter channel (FSC) photodiode and provides information on the relative size of the particle. Light measured at a 90° angle to the laser excitation beam is collected by the Side Scatter channel (SSC) photomultiplier tube (PMT) and provides information on the relative granularity of the particle.

Separate fluorescence channels (FL-) detect any fluorescent light associated with the particle. The light is directed via a series of mirrors and filters to PMT´s. Filters determine the wavelength of light to be detected by the PMT, any unfiltered light is reflected by a dichoric filter and is channelled to the next fluorescent channel.

Fluidics System

The fluidics system is essential for transporting the particles from a random mix into and orderly stream of single file particles.

The point at which the particles are delivered into the sheath fluid is commonly termed the Flow Cell. The sample is injected into the core of the sheath fluid stream, which then accelerate and pass through a narrowing channel, channelling the particles in the core of the sheath fluid into single file. This phenomenon is known as hydrodynamic focusing and presents a single file of particles to the laser/s.

Electronics

Light hits the photodetector and a small current is generated, the associated voltage of which is amplified and then represented by electrical signals via an analogue to digital converter. These electrical signals are what are seen on the computer connected to the Cytometer.

Cell Sorting

Populations of highly purified cells with specific characteristics can be separated by flow cytometers capable of cell sorting. Sort decisions can be made based on relative size and granularity of cells alongside fluorescence associated with them.

Cells pass through the lasers in a chargeable sheath fluid and a decision is made as to whether the cells are required for sorting. The stream is then passed through a narrow rapidly vibrating orifice, this causes the stream to break-off into droplets, cells are captured in these droplets at this droplet-breakoff point.

The time between the cells passing through the laser and droplet-breakoff is stably maintained, therefore the time between seeing the desired cell and it´s encapsulation in a droplet is known, and the appropriate droplet containing the cell of interest can be charged. The droplets pass through high-voltage deflection plates and charged droplets are deflected appropriately into a collection vessel, uncharged droplets enter the waste.

Please contact a member of the Flow Cytometry staff for information should you wish to undertake cell sorting

Filters and configurations 

 

FACSAria IIu

Violet laser 355nm

Violet 450/40

Violet 525/50

Blue laser 488nm

Blue 530/30

Blue 575/26

Blue 610/20

Blue 660/20

Blue 695/40

Blue 780/60

Red laser 633nm

Red 660/20

Red 730/45

Red 780/60

FACSCalibur

488nm laser

FL1 = 530/30

FL2 = 575/26

FL3 = 650LP

633nm laser

FL4 = 650LP

LSRII

UV laser 355nm

UV 450/50

UV 530/30

Violet laser 405nm

Violet 450/40

Violet 525/50

Blue laser 488nm

Blue 530/30

Blue 575/26

Blue 610/20

Blue 695/40

Blue 780/60

Red laser 633nm

Red 660/20

Red 780/60

Aurora

V1 – 428

V2 – 443

V3 – 458

V4 – 473

V5 – 508

V6 – 525

V7 – 542

V8 – 581

V9 – 598

V10 – 615

V11 – 664

V12 – 692

V13 – 720

V14 – 750

V15 – 780

V16 – 812

B1 – 508

B2 – 525

B3 – 542

B4 – 581

B5 – 598

B6 – 615

B7 – 661

B8 – 679

B9 – 697

B10 – 717

B11 – 738

B12 – 760

B13 – 783

B14 – 812

R1 – 661

R2 – 679

R3 – 697

R4 – 717

R5 – 738

R6 – 760

R7 – 783

R8 – 812