Flow cytometry core service
The Medical School's flow cytometry facility is a core facility based in the RHH Medical School and provides a service to all at the University and externally.
New arrival: Nanoanalyser
The nanoanalyser can detect and quantify small particles from 40-1000nm (such as extracellular vesicles, polymer particles and lipid nanoparticles), and can detect two fluorescent properties
Background
Flow cytometry is a technique that enables single whole particles/cells to be analysed. Events pass through lasers and physical properties such as relative size (FSC) and granularity/internal complexity (SSC) are detected. If there is any fluorescence also associated with the cell this can also be detected. The NanoFCM and FACSMelody detect this fluorescence with the emitted light being diverted via a series of mirrors and filters and collected by PMTs with specific bandpass filters in front of them.
Phycoerthrin (PE) - Excitation Max 561 nm
- Excitation 488nm laser
- Detection Band pass filter 575/26nm (ie 562-588nm)
The Aurora cytometers use spectral technology. This technology allows the full spectrum of fluorochrome emission to be detected across multiple lasers. This increases the sensitivity of detection and enables many more fluorochromes to be detected and separated, up to 40, compared to 鈥渃onventional鈥 flow cytometry.
Services provided by the facility
Analysis
Single-cell/particle suspensions can be stained with fluorescent proteins/dyes/antibodies to undertake various assays on the analytical flow cytometers (3 and 5 laser Aurora).
- Immunophenotyping (up to 40 colours on Cytek Aurora 5 laser)
- Cell cycle
- Viability/Apoptosis/Necrosis
- Calcium flux
- Cell counts
- Detection of Extracellular vesicles, bacteria, algae
- Transfection efficiency (e,g GFP, mCherry)
Small particle analysis
The newest addition to the Core Facility is a NanoFCM, nanoanalyser. The nanoanalyser is capable of detecting small particles, from 7-1000nm, compared to the Aurora鈥檚 which can detect particles sized from 100nm. There are two fluorescent detectors on this instrument, with the option of 488nm or 640nm excitation for the second detector.
- extracellular vesicles
- mitochondria
- bacteria
- viruses
- Lipid nanoparticles (LNP)
- Gold particles
- nanomaterial
Cell sorting
Using physical or fluorescent properties, cells can be separated from a parent population into different tubes. This is called cell sorting. Cells can be bulk sorted into tubes, or single cells can be sorted into 96/384 well plates, this work can be undertaken on BDFACSMelody.
Fluorochrome choice and experimental design
Before coming to the facility, we advise that you contact facility staff regarding which machine is most appropriate, fluorochrome choice, antibody titration and controls required for correct instrument settings. There is an enquiry form further
Training
Facility staff can run samples for users, or users can be trained to set up and run samples unassisted on all the machines.
All users must undertake three training sessions, provide a COSHH number, read risk assessments and complete the sign-off sheet before running samples unassisted.
Analysis
Facility staff can provide advice and support users in analysing the data generated in the facility. All users can have access to analysis software DeNovo FCSExpress software for a nominal annual charge.
Equipment available
Aurora 3 laser
405 (violet), 488 (blue) and 633 (red)nm lasers.
Any 12x75mm tube and 96, 384 well plates can be loaded
Violet 405nm
16 channels (420-829nm)
Blue 488nm
14 channels (498-829nm)
Red 633nm
8 channels (652-829nm)
Aurora 5 laser
355 (UV), 405 (violet), 488 (blue), 561 (yellow/green)and 633 (red) nm lasers.
Any 12x75mm tube and 96, 384 well plates can be loaded
UV 355nm
10 channels 365-829nm
Violet 405nm
16 channels 420-829nm
Blue 488nm
14 channels 498-829nm
Yellow-Green 561nm
10 channels 567-829nm
Red 640nm
8 channels 652-829nm
Nanoanalyser (small particle)
488 (blue) and 633 (red)nm lasers.
0.5 ml eppendorfs loaded only, provided by the flow facility
Filters and configuration
Blue 488nm
Blue 525/40
And Blue 488nm/Red 640nm
Blue 580/40 OR Blue 670/30 OR Blue 710/40 OR Red 670/30 OR Red 710/40
FACSMelody (cell sorter)
488 (blue), 561 (yellow/green) and 633 (red)nm lasers.
BDFalcon tubes loaded only, provided by the flow facility
Blue laser 488nm
527/32
700/54
Yellow/green 561nm
582/15
613/18
697/58
783/56
Red laser 640nm
660/10
783/56
Bookings and charges
Technical time can be booked on the Flow Google Calendar and all the machines can be booked through the Flow Lab on Clustermarket. Please contact the facility for access.鈥嬧嬧嬧
Contact us at flow@sheffield.ac.uk.
Analysers
Aurora 3-laser or 5-laser: 拢55 per hour
Sorter
FACSMelody - 拢80
Filters and configurations
- FACSMelody
-
Blue laser 488nm
527/32
700/54
Yellow/green 561nm
582/15
613/18
697/58
783/56
Red laser 640nm
660/10
783/56
- Aurora (3-laser)
-
Violet 405nm
16 channels (420-829nm)
Blue 488nm
14 channels (498-829nm)
Red 633nm
8 channels (652-829nm)
- Aurora (5-laser)
-
UV 355nm
10 channels 365-829nm
Violet 405nm
16 channels 420-829nm
Blue 488nm
14 channels 498-829nm
Yellow-Green 561nm
10 channels 567-829nm
Red 640nm
8 channels 652-829nm