Flow Cytometry Core Service
The Medical School Flow Cytometry Core Facility is a resource available for all within the University, able to undertake work for external groups too. The facility comprises of 3 analysers, 2 cell sorters, soluble cytokine quantification service, FlowJo and FCSExpress analysis software licenses.
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.
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
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BDFACSCalibur – 488 and 633nm lasers. 4 detectors. Tube loading only.
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BDLSRII – 355, 405, 488 and 633nm lasers. 13 detectors. Tube loading only.
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Cytek Aurora – 405, 488 and 633nm lasers. 38 detection channels. Tube and plate loading.
Sorters
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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)
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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.
Equipment booking
Technical time can be booked on the flow@sheffield.ac.uk google calendar and all the machines can be booked through the Flow Lab on Clustermarket. Please contact the facility for access.
- Bookings and charges
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Bookings, charges and contact details
All bookings with Technical Support - £100 per hour
Charges from September 2021
Analyser
BDFACSCalibur - £30 per hour
BDLSRII - £45 per hour
Cytek Aurora - £55 per hour
Sorter
BDFACSAria - £100 per hour (always require Technical Support)
BDFACSMelody - £80 unassisted
Staff and contact details
Sue Clark – Flow Facility Lead Technician. Working days Mon, Tues, Thurs and Friday
Kay Hopkinson – Flow Facility Senior Technician Working days Weds
Jess Medcalf – Research Technician
Eva Wild – Research Technician
Email – flow@sheffield.ac.uk
Room DU27
D Floor
Medical School
Beech Hill Road
Sheffield
S10 2RX
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
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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
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488nm laser
FL1 = 530/30
FL2 = 575/26
FL3 = 650LP
633nm laser
FL4 = 650LP
- LSRII
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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
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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