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.
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 seen, 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
- Emission Max 576nm
- Detection Band pass filter 575/26nm (ie 562-588nm)
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.
Example applications
- 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)
Services provided by the facility
The facility comprises
- 2 Cytek Aurora spectral analysers
- 1 LSRII analyser
- 1 FACSMelody cell sorter
- A soluble cytokine quantification service
- Analysis software FCSExpress (available from August 2023)
Unlike the traditional flow cytometers which detect only a small portion of a fluorochromes spectral emission, the spectral analysers collect all the light emitted. This enables multiple fluorochromes to be separated and analysed simultaneously.
In May 2023 we took delivery of a second Cytek Aurora. This is a five-laser system with a plate loader capable of detecting up to 40 different colours.
Our staff can help you with all aspects of your work including planning and running experiments and data analysis.
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.
Contact us at flow@sheffield.ac.uk.
Analysers
LSRII: £45 per hour
Aurora 3-laser or 5-laser: £55 per hour
Sorter
FACSMelody - £80
Filters and configurations
- 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 660/20
Blue 695/40
Blue 780/60
Red laser 633nm
Red 660/20
Red 730/45
Red 780/60
- 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