Flow Cytometry Core Service

The Medical School Flow Cytometry Core Facility is a resource available for all within the University, as well as undertaking work for external groups and comapnies. The facility comprises of 4 analysers, 2 cell sorters, a soluble cytokine quantification service and FlowJo analysis software licenses.

Services provided by the facility

Experimental design

Advice regarding controls, dyes, fluorochrome choice, cell number and staining protocols

Training

Users can undertake a 3 session training course, and can then be signed off to run samples independently on the analysers and the FACSMelody cell sorter. For an extra fee, samples can be processed for users by Flow Cytometry staff

Analysis

Advice can be provided to assist with data analysis, using both the machine's acquisiton-analysis software, or FlowJo analysis only software


Machines available

Analysers

  • 2 x FACSCalibur - 2 lasers 4 detectors. Tube loading only.
  • Attune Autosampler - 2 lasers 6 detectors. Tube and 96 well plate
  • LSRII - 4 lasers 13 detectors. Tube loading only.

Sorters

  • FACSMelody - 3 lasers 8 detectors. Tube loading only, can sort up to 2 populations simultaneously
  • FACSAria IIu - 3 lasers 9 detectors. Tube loading only, can sort up to 4 populations simultaneously

BDFACSMelody

The flow cytometry core facility is delighted to announce that they have taken delivery of a new cell sorter, the BDFACSMelody. This machine has 3 lasers with 488, 561 and 633nm wavelengths and 8 fluorescent detectors. Cells can be sorted into a variety of collection devices including eppendorf, BDFalcon 12x75mm tubes and 96 well plates.

Miscellaneous

  • Countess - automated cell counter
  • gentleMACS Dissociator - automated tissue dissociator separating organs and tissues into sterile single cell suspensions

Equipment booking

Bookings and charges

Bookings, charges and contact details

Bookings

Charges from April 1st 2019

ANALYSERS
Attune
 £30
With technical support £80

FACSCalibur and FACSCalibur New £28
With technical support £80

LSRII £37
With technical support £80

CELL SORTERS
FACSAria 
Always with technical support £80

FACSMelody  £60
With technical support £80

CBA Assays- Contact Flow Cytometry Core Facility for quotes.

FlowJo Analysis Computer
Free to use

There are also numerous remote computers on which to perform analysis and for a small annual charge the FlowJo software is available to download onto Mac or PC.

Contact Details for Bookings

flow@sheffield.ac.uk

Telephone:  Lab - 0114 215 9084 Office - 0114 215 9213

Address:
Room DU32
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 

Attune Configuration

488nm laser

BL1 = 530/30

BL2 = 574/26

BL3 = 690/50

BL4 = 780/660

633nm laser

RL1 = 660/20

RL2 = 780/60

FACSAria IIu

Violet laser 355nm

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

FACSCalibur (old and new)

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

LSRII Filters and suggested fluorochromes

UV laser 355nm

UV 450/50

Phenotyping

BDHorizon BUV 395*

AlexaFluor 350

Fixable viability

Fixable viability dye eFluor455 UV

Zombie UV Fixable Viability Kit

Blue LIVE/DEAD Fixable Dead Cell Stain

Viability/cell cycle

Hoeschst333422

Hoeschst333258

Hoeschst345580

DAPI

 

UV 530/30

Phenotyping

BDHorizon BUV 496*

 

Fixable viability

Aqua LIVE/DEAD Fixable Dead Cell Stain

 

Viability/cell cycle

DAPI

 

Extra filters that can be swapped in

BP 670/14 BDHorizon Brilliant UV 661*

BP720/40 BDHorizon Brilliant UV 737*

No filter currently available in Flow Lab BDHorizon Brilliant UV 805*

 

*BDHorizon Brilliant –Stain Buffer must be used when more than 1 Brilliant dye is present in a panel

 

Violet laser 355nm

Violet 450/50

Phenotyping

BD Horizon BV421

BD Horizon BV450

Pacific Blue

Alexa Fluor 405

Cascade Blue

eFluor 450

 

Fixable viability

Zombie Violet Fixable Viability Kit

Violet LIVE/DEAD Fixable Dead Cell Stain

 

Viability/cell cycle

Hoeschst333422

Hoeschst333258

Hoeschst345580

DAPI

 

Violet 525/50

Phenotyping

BDHorizon BV500*

BDHorizon BV510*

Cascade Yellow

Alexa Fluor 430

Pacific green

Fixable viability

Yellow LIVE/DEAD Fixable Dead Cell Stain

 

Viability/cell cycle

DAPI

 

Blue laser 488nm

Blue 530/30

Phenotyping

FITC

BD Horizon BB515

Alexa Fluor 488

 

Fixable Viability

Blue LIVE/DEAD Fixable Dead Cell Stain

Zombie Green Fixable Viability Kit

 

Blue 575/26

Phenotyping

PE 

BD Horizon BV605

 

Viability/Cell Cycle

PI

 

Blue 610/20

Phenotyping

BD Horizon 605

BD Horizon PE-594

PE-Texas Red

PE-Alexa Fluor 610

PE-eFluor 610

Viability/Cell Cycle

PI

 

Blue 660/20

Phenotyping

PECy5

PerCp

Viability/Cell Cycle

PI

7AAD

 

Blue 695/40

Phenotyping

PerCp

PerCp Cy5.5

PE Cy5.5

PE-Alexa Fluor 680

PerCp eFluor 710

 

Blue 780/60

Phenotyping

PECy7

PE Alexa Fluor 700

 

Red laser 633nm

 

Red 660/20

Phenotyping

APC

Alexa Fluor 647

eFluor 660

Fixable viability

Zombie Red Fixable Viability Kit

Far Red LIVE/DEAD Fixable Dead Cell Stain

 

Viability/cell cycle

TOPRO3

 

Red 730/45

Phenotyping

BDHorizon APC700

Alexa Fluor 700

 

Red 780/60

Phenotyping

APCCy7

APCH7APCeFluor780

 

Fixable viability

Zombie Near Infa Red Fixable Viability Kit

Near Infa Red LIVE/DEAD Fixable Dead Cell Stain

Filters can be changed and new ones ordered should users wish to use dyes/fluorochromes that can not be detected with the current configuration

This list is a guide, you must run your samples with appropriate controls to ensure your panel works correctly

Suppliers

Biolegend

BD Bioscience

Beckman Coulter

Life Technologies

eBioscience

Spectrum viewers

Invitrogen

BDBioscience

BioLegend

eBioscience

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