Core Technologies
Fluorescence Cytometry
We recommend that before purchasing a new fluorophore you check to see if it is detectable with our current filter sets.
Accessory instrumentation
We have two high throughput sampler modules for use with the fortessa type systems and symphony A5 capable of automated running of multiwell plates.
Help and training
The facility has a particular emphasis on flow cytometry education. We deliver both basic and advanced flow cytometry training.
We can offer help and assistance to all users. We can help you in experimental set up, fluorochrome choice or any other issues that impact on the use of the facility.
If you have any questions please contact us.
Cell Sorters
Isolate and recover your cells using our cell sorter systems. Our cell sorters are:
- BD FACS Aria IIu (Centre for Life)
- BD FACS Aria III (NICR Herschel Building)
- BD FACS Fusion (Medical School)
- MoFLO Astrios (Medical School)
We also have information and support on using our cell sorters
All four of our sorters are in containment hoods allowing us to sort unscreened human material.
| Laser | Bandpass Filter | Suggested Flurochromes |
|---|---|---|
| Blue 488nm |
530/30 710/50 |
Brilliant Blue 515, FITC, Alexa Fluor 488, GFP, YFP, Zombie Green PerCP-Cy5.5 |
| Yellow/Green 561nm |
582/15 610/20 710/50 780/60 |
PE PE-Dazzle, PE-CF594 PE-Cy5.5 PE-Cy7 |
| Red 635nm |
670/30 730/45 780/60 |
APC, Alexa Fluor 647 Alexa Fluor 700 APC-H7, APC-Cy7, DRAQ5, Zombie NIR |
| Violet 405nm |
450/50 525/50 610/20 670/30 710/50 780/60 |
BV421, V450, DAPI, Pacific Blue BV510, Zombie Aqua, V500 BV605 BV650 BV711 BV786 |
| UV 355nm |
379/28 450/50 730/45 |
BUV395 DAPI, Hoechst blue BUV737 |
Our BD FACSAria systems have identical configurations across 5 lasers. They are capable of sorting into: tubes, culture plates, and microscope slides. They can sort up to 4 populations simultaneously at high speed.
Our most advanced analyser, the BD Symphony A5 is based at our medical school site. With 5 lasers and 28 different fluorescent detectors the Symphony can be utilised for highly complex panels. The instrument configuration is shown below.
| Laser | Bandpass Filter | Suggested Flurochromes |
|---|---|---|
|
Blue 488nm
|
530/30 610/10 670/30 695/40 780/60 |
Brilliant Blue 515, FITC, Alexa Fluor 488, GFP, YFP, Zombie Green
PerCP BB700, PerCP-Cy5.5 |
| Yellow/Green 561nm |
586/15 610/20 670/30 710/50 780/60 |
PE PE-CF594, PE-Dazzle, Propidium lodide PE-CY5 PE-CY5.5 PE-Cy7 |
| Red 635nm |
670/30 730/45 780/60 |
APC, Alexa Fluor 647 Alexa Fluor 700 APC-H7, APC-Cy7, DRAQ5, Zombie NIR |
| Violet 405nm |
450/50 525/50 586/15 610/20 670/30 710/50 750/30 780/60 |
BV421, V450, DAPI, Pacific Blue BV480, BV510, Zombie Aqua, V500
BV605 BV650 BV711 BV750 BV786 |
| UV 355nm |
379/28 515/30 580/20 605/20 670/30 740/35 820/60 |
BUV395 BUV496 BUV563
BUV661 BUV737 BUV805 |
We have two advanced Fortessa style systems, one at each of our main sites. Our Fortessa X20 system is based at the medical school facility and our Fortessa system based at the Centre for Life. Both instruments share a matched configuration allowing panels designed for one to be easily analysed on the other.
The laser and filter configuration is shown below, All the fluorochromes listed in the tables have been validated to work on our instrumentation individually or as part of multi colour panels.
| Laser | Bandpass Filter | Suggested Flurochromes |
|---|---|---|
| Blue 488nm |
530/30 710/50 |
Brilliant Blue 515, FITC, Alexa Fluor 488, GFP, YFP, Zombie Green PerCP-Cy5.5 |
| Yellow/Green 561nm |
582/15 610/20 710/50 780/60 |
PE PE-Dazzle, PE-CF594 PE-Cy5.5 PE-Cy7 |
| Red 635nm |
670/30 730/45 780/60 |
APC, Alexa Fluor 647 Alexa Fluor 700 APC-H7, APC-Cy7, DRAQ5, Zombie NIR |
| Violet 405nm |
450/50 525/50 610/20 670/30 710/50 780/60 |
BV421, V450, DAPI, Pacific Blue BV510, Zombie Aqua, V500 BV605 BV650 BV711 BV786 |
| UV 355nm |
379/28 450/50 730/45 |
BUV395 DAPI, Hoechst blue BUV737 |
Located in the Paul O'Gorman building, the Attune NxT has 2 lasers (Blue 488nm and Violet 405) and up to 8 fluorescent parameters. The Attune boasts new technology in the form of acoustic focusing, allowing high flow rates whilst maintaining tight fluorescent populations.
More information about the Attune NxT can be found at Thermo Fisher Scientific
The Attune NxT also comes ready for 96 well plate analysis with seperate autosampler. The config for the Attune NxT is shown below.
| Laser | Bandpass Filter | Suggested Fluorochrome |
|---|---|---|
| Blue 488 |
530/30 574/26 695/40 780/60 |
Brilliant Blue 515, FITC, Alexa Fluor 488, GFP, YFP, Zombie Green PE PerCP Cy-5.5 PE-Cy7 |
| Violet 405 |
440/50 512/24 603/48 710/50 |
BV421 V450, DAPI, Pacific Blue BV510 Zombie Aqua, V500 BV605 BV711 |
The Attune NxT is located in the Herschel building with 3 lasers (Blue 488nm Red 637nm and Violet 405nm) and up to 11 fluorescent parameters. The Attune boasts new technology in the form of acoustic focusing, allowing high flow rates whilst maintaining tight fluorescent populations.
More information about the Attune NxT can be found at Thermo Fisher Scientific
The config for the Attune NxT is shown below.
| Column 1 | Column 2 | Column 3 |
|---|---|---|
|
Blue 488nm
|
530/30 574/26 695/40 780/60 |
Brilliant Blue 515, FITC, Alexa Fluor 488, GFP, YFP, Zombie Green PE PerCP Cy-5.5 PE-Cy7 |
| Red 635nm |
670/30 730/45 780/60 |
APC, Alexa Fluor 647 Alexa Fluor 700 APC-H7, APC-Cy7, DRAQ5, Zombie NIR |
| Violet 405 |
440/50 512/24 603/48 710/50 |
BV421 V450, DAPI, Pacific Blue BV510 Zombie Aqua, V500 BV605 BV711 |
Mass Cytometry
Use cutting edge technology to get more from your samples.
Helios
Mass cytometry with Helios allows the high dimension analysis of single cells using CyTOF technology. Designed to significantly reduce signal overlap, mass cytometry allows the simultaneous interrogation of 40+ markers of interest tagged by antibodies labelled with rare earth metals.
The Helios is the latest generation of the Cytof Mass Cytometer combining a flow cytometer with the power of a Time of Flight mass spectrometer
The Helios is the latest generation of the Cytof Mass Cytometer. These systems combine the sample delivery and data output of a flow cytometer with the detection capabilities of a Time of Flight mass spectrometer.
The technology is conceptually akin to traditional fluorescent flow cytometry except that antibodies and detection reagents are tagged with stable isotopes of rare earth metals instead of fluorochromes. The minimal overspill allows for highly multiplexed assays to be performed producing high dimensional single cell data.
Capabilities
- Simultaneous detection of over 40 cellular markers including surface antigens, transcription factors and cytokines. Plus DNA markers, multiplexed sample barcoding and internal QC bead normalisation
- Lessened spill over compared to fluorescent cytometry. Compensation is not required
- Analysis of fixed and cryopreserved samples
Service
We encourage users to contact us in the early planning stages of mass cytometry experiments. We offer advice on antibody panel design, sample preparation, and conjugating antibodies if they are not commercially available. For full details on these and all our other services see here
Core staff will acquire the samples and export the data (FCS files) for your independent analysis or in conjunction with a bioinformatician. Please book using our usual booking resource.
Considerations
We have optimised protocols for immunophenotyping using mass cytometry:
- All samples must be fixed and stable in dH2O
- All samples will be run at 0.5 x106 cells/mL and filtered through a 40 micron filter. “EQ” normalisation beads will be added to your sample to a final concentration of 10% v/v.
- It takes around 70 minutes to acquire 1 million cells. This is at a fixed sample introduction rate of 30 uL/min.
Pricing can be found on our prices page.
Hyperion
The Hyperion Imaging System brings CyTOF technology together with imaging capability to allow the simultaneous detection of up to 37 different markers using Imaging Mass Cytometry™ (IMC™). Using the Hyperion allows analysis of tissue architecture and spatial relationships producing high dimensional image data from tissue sections.
The Hyperion Tissue Imaging unit allows for high dimensional analysis of tissue sections by mass cytometry. Tissue is analysed directly from the slide; preserving spatial relationships and eliminating the need to disassociate into single cell suspensions.
Imaging is achieved through laser ablation of sections stained with isotope-labelled antibodies and probes. Clouds of these rare earth metals are analysed by time of flight (TOF) cytometry and digitally reconstructed for in-depth image analysis.
Capabilities
Simultaneous detection of over 40 cellular markers throughout tissue sections. These include phenotypic antigens, structural markers, transcription factors and cytokines.
- Lessened spill over compared to fluorescent imaging approaches.
- Analysis of FFPE or Fixed-Frozen tissue sections.
- Multi-dimensional image and singe cell analysis through cell-segmentation including neighbourhood analysis
Service
It is essential to contact us in the early planning stages of imaging mass cytometry experiments. We offer advice on antibody panel design, sample preparation and conjugating antibodies if they are not commercially available.
You will work with the core staff to identify regions of interest (ROI) for ablation. Core staff will then ablate the ROI and export the data (MCD files) for your independent analysis or in conjunction with a bioinformatician. Please book using our usual booking resource.
Considerations
Please contact us in the planning stages of all IMC experiments.
Pre-validation of antibody clones is essential to ensure they work on tissue sections. Immunofluorescence without secondary amplification is most comparable to intensities observed with imaging mass cytometry.
Sections should be between 4-12 micron thickness, mounted on normal glass slides (approximately 7.5 x 2.5cm) and should not have a coverslip. Please contact us for guidance on the ablatable area of a slide.
Multiple tissue sections per slide increases the throughput of a project.
We recommend regions of interests not to exceed 1mm2, however multiple ROI per section is possible. It takes around 3h to ablate 1mm2.
Control tissue sections are strongly advised.
Pricing
Costs are determined by the duration of time for which the instrument is required. Up to date rates are found on our prices page
Metabolimetry
Assess the metabolic activity of your cells with our Seahorse XF analyser systems
Seahorse XF Analyser
The newest addition to the FCCF, Agilent Seahorse XF technology measures cellular metabolism in live cells.
Living cells use one or both of the major energy producing pathways:
- mitochondrial respiration
- glycolysis
Seahorse XF technology measures cellular metabolism in live cells in real time.
Seahorse XF Technology
Solid-state sensors measure extracellular flux (oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)) providing insight into mitochondrial function and glycolytic activity.
Seahorse XF sensors simultaneously measure both OCR and ECAR in every well of either 96 (Seahorse XF96 system) or 24 well plates (Seahorse XF24 system). All platforms support injection of up to 4 unique compounds per well. At FCCF we have one XF96 at the medical school and one XF24 instrument at the Health Innovation Neighbourhood.
Imaging Cytometry
ImageStream X Mark II
Here at the FCCF we have access to an ImageStream X Mark II system. The ImageStream provides unrivalled sensitivity combining the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy. The use of a high definition CCD Camera as a detector provides a novel and unique look at your cells.
Newcastle FCCF has a wealth of experience using this system to produce high quality publications and novel method development research.
To learn what imaging flow cytometry can offer your research please take a look at a presentation from the FCCF director Andrew Filb.
Spectral Cytometry
The Aurora 5L and The Northern Lights
Information about both the Aurora and Northern Lights systems can be found on the Cytek website.
For panel design assistance please see the Cytek Spectral Viewer