ISAC Cyto 2019 Vancouver Highlights

At the end of May, Bee and Chris traveled to Vancouver to attend the annual International Society for the Advancement of Cytometry (ISAC) Congress. Below are the highlights of the meeting which we would like to share with you.

 

Tuneable fluorophores: During the innovations session two companies showed off their concept of tuneable fluorophores. The idea being that you can alter the brightness of the fluorophore to match the expression level of the antigen being analysed. In theory, by doing this you can choose well separated fluorophores without worrying about brightness, allowing improved data resolution.  Both use long chains of fluorescent molecules, where adjusting the length changes their brightness. The difference is in their construction; Phitonex uses DNA scaffolds to hold the molecules, whereas StabiLux use Boron Nitride nanotubes.

Activome: Katarzyna Groborz from Marcin Poręba’s group at the Wroclaw University of Technology discussed their concept of the activome (an –ome that sits on top of the genome, epigenome, transcriptome etc.) through the use of activity based probes directed at enzymes. These probes, when conjugated to metal isotopes for detection by mass cytometry (Helios and Hyperion systems), allows the current active metabolic processes to be probed in the cell.

Optogenetics: Is the process of using light to control biological processes in cells. There is a nice overview in Max-Planck Gesellshaft: https://www.mpg.de/18011/Optogenetics. During the innovation session Opto Biolabs showed us their flow cytometer add-on that allows real-time analysis if optogenetic processes on the flow cytometer. They are currently looking for trial partners (beta testers).

Microbubbles: Akadeum were showing off their novel cell isolation kits that use microbubbles to separate cells. The concept is the same as the Invitrogen, StemCell, and Miltenyi magnetic bead isolation kits, but using buoyant bubbles.  They have developed a way to make these bubbles stable over long periods and to attach antibodies to them.  This way you don’t require a magnet or column to separate your cells.  There is a sample vial on Chris’ desk if you would like to see them.

opt-SNE: Anna Belkina talked about her preprint in bioRxiv detailing a method to optimise parameter choice when using tSNE dimensionality reduction, especially useful for larger datasets.  The paper goes into some detail about the effect of various parameters and how to calculate when you have reached the “ideal” iteration.  opt-SNE is available in the latest version of FlowJo and at this link: Omiq.

 

FAUST:  There were two posters from the authors of the FAUST (full annotation using shape-constrained trees) algorithm for discovering and annotating populations.  They take the direction of “standardizing and partitioning the space across samples and using the discovered phenotypes to match discovered cell populations across independent samples”.  They subsequently used this to discover novel biomarkers in previously published studies.  The preprint is here: https://www.biorxiv.org/content/10.1101/702118v1 and the code is here: https://github.com/RGLab/FAUST

 

metaCyto: Atul Butte from the University of California discussed his groups’ tool that allows the comparison and combination of mass and fluorescence cytometry data.  metaCyto clusters both types of data together using silhouette scanning.

Berkley lights: The Beacon has been out for a while, but it has now be supplemented with a significantly cheaper lower throughput model called the Lightning, aimed at research labs.  If you have not seen Berkley Light’s machines then watch this video now.  They use light to move individual cells into pens, where they can grow, be stimulated, identified, and moved back out at the touch of a button.

REAlease antibodies: Miltenyi released these late last year.  They are antibodies that can be fully removed from the cell after use.  The idea being you can remove them after sorting so they don’t interfere with downstream processing and cell culture, or they can be easily removed for multiplexed sequential image analysis.

REAfinity antibodies: Miltenyi has also geared up in making high quality antibodies that offer superior lot-to-lot consistency and purity as compared to conventional mouse or rat monoclonal antibodies. They have been engineered to lack any background binding and also with the same human IgG1 isotype, eliminating the need to include multiple isotype controls during flow analysis.

 

Spectral analysers: Sony launched their new spectral analyser, the ID7000.  This instrument uses 188 detectors to analyse the spectrum of a fluorophore to allow the separation of similar dyes that would not be possible on a traditional flow cytometer; such as GFP and YFP or APC and AF647.  There are two spectral analysers currently on the market, the other being the CytTek Aurora.

 

Advancement in Imaging flow cytometry

High speed Imaging: Hideharu Mikami from Goda’s Lab has developed a novel way to increase the throughput of fluorescence imaging flow cytometry (FIFC) by virtual-freezing of flowing cells and spatiotemporal control of excitation beam illumination to allow image acquisition with a high throughput of ~10,000 cells/sec without compromising imaging sensitivity.

 

Image free ‘Imaging’ flow cytometry: Toru Imai from Thinkcyte Inc., has make a recent development and advancement for an ultrafast fluorescence ghost ‘imaging’ activated cell sorter (FiCS). This work is being carried out by integrating a microfluidic cell sorting platform with high throughput cell classification based on supervised machine learning algorithm.

High speed Imaging                                                   Image free ‘Imaging’ flow cytometry

 

 

 

 

 

 

 

 

Invitation to Contribute to the Special Issue on Genomic Cytometry in Cytometry, Part A

On behalf of Editor-in-Chief, Dr. Attila Tárnok, we would like to invite you to submit a manuscript for the Special Issue of Cytometry, Part A focused on Genomic Cytometry.

Recent advances in the ability to deeply characterise individual cells have been made possible with technologies that have generally been lumped under the heading of “single cell genomics”. The term itself however is potentially misleading. As these new techniques are designed to measure cells, whether this be at the protein, RNA, DNA or DNA conformation level, at the resolution of the single cell, we believe these techniques should be considered under the banner of Genomic Cytometry. Here we define genomic cytometry in two ways:

  • Genomic Cytometry is no different to standard cytometry except it uses a genomic readout (base pair sequences from created libraries) rather than fluorescence, mass or imaging.
  • Genomic Cytometry is the measurement of cells using nucleic acid base pair sequence as reporters. Whether these reporters are inherent in the cell – i.e. RNA/ DNA sequence or if known base pair sequences (genomic barcodes) are surrogates for other measures – i.e. unique molecular barcodes attached to antibodies or regions of conformational change, these techniques should be considered under the banner of Genomic Cytometry.

This Special Issue on Genomic Cytometry provides a valuable forum where scientists, engineers, developers and shared resource laboratory manager will be able to share their most recent findings and discuss challenges in the field. In addition to articles clearly explaining basic principles, advanced techniques, and applications of genomics cytometry. We are looking for clear and concise reviews of the current state of the field (particularly in relation to Cancer, Immunology, Development and non-Mammalian systems) as well as perspective from key opinion leaders as to the current state and future of this rapidly emerging field.

We have structured the issue to cover all aspects of Genomic cytometry and are seeking articles in the following areas:

Technology

  • Single cell transcriptomics
  • Single cell DNAseq
  • Single cell epigenetics
  • Oligo antibodies (CiteSeq, totalSeq, Abseq)
  • Imaging genomic cytometry
  • Combinatorial approaches – Multi-omics
  • Technology for clinical applications
  • Informatics analysis platforms
  • Perspective review on technology and where it needs to be for wide adoption in research and clinical application.
  • Associated technologies – microfluidics, gentle cell sorting, rapid enrichment and cell counting, automated sample prep etc.

Assays

  • Any new assays in genomic cytometry
  • Any new assays associated with Genomic cytometry – either upstream in sample prep or downstream in validation process

Biology

  • Cancer
  • Immunology
  • Developmental
  • Non-mammalian
  • Normal function and Human cell atlas

Associated technologies and how they are used in genomic cytometry workflow

  • Suggest a combined paper discussing the application of fluorescence and mass cytometry to single cell genomics/genomic cytometry assays as part of the validation approach.

Shared Resource Laboratory

  • A SRL perspective for Genomic cytometry

The target submission date is Sept 30th 2019 with a publication date in March 2020 (with the possibility of an extension until Oct 31st with a publication date in July 2020). All manuscripts will be peer-reviewed, following standard practices of Cytometry, Part A. Each individual article of the special issue will be published online as soon as it is accepted.

For more information on Cytometry, Part A with regard to author guidelines and publication charges, please, refer to the journal website at https://onlinelibrary.wiley.com/journal/15524930. When ready, please submit your manuscript directly at the Cytometry, Part A manuscript submission site. Please state in your cover letter that the submission is intended for this Special Issue.

Guest Editors

Robert Salomon

David Gallego-Ortega

Christopher Hall

July 2019

July marks the introduction of our “application of the month” which will be a reoccurring segment highlighting interesting and relevant applicational notes on particular flow cytometric techniques. Other noteworthy mentions this month concern the MA900, iGrow training, the upcoming MACC meeting, what the Mo-Flo XDP cell sorter can offer you, sheath fluid, and cytokine analysis.

Downtime

All staff operated sorters are unavailable Monday mornings, from 10:00am-11:00am to accommodate a staff meeting. All machines are unavailable on the morning of Tuesday 9th July for a routine deep clean. The Sony’s are unavailable on alternating fortnightly Tuesday mornings for their routine cleans. For up to date instrument availability, please visit our PPMS online booking calendar.

Sony MA900

The MA900 is now live with users having the ability to book the machine with staff assistance. To learn how to operate the machine independently, you can sign up on iGrow. Users who were previously trained on the SH800 can re-attend the training session to be made familiar with changes in both the hardware and software, or can make an assisted booking on the machine and have a member of staff be present to detail these changes in person.

MACC Meeting

Each quarter the Mid Anglian Cytometry Club (MACC) meets to discuss science, trends in cytometry, techniques, and to network with one another.  The next meeting is on Wednesday 17th July 1800-2200 at Sanger (C302).  The program includes talks by Sanger staff; Bee Ling Ng and Annie Speak, and news from the latest Cytometry Conference in Vancouver from Rachael Walker (Babraham) and Mateusz Strzelecki (CRUK). All are welcome to attend.

Beckman Coulter Mo-Flo XDP

The Mo-Flo XDP is a 5 laser cell sorter with up to 15 detectors and a highly customisable optical filter pattern to accommodate complex fluorophore panels. The sorter boasts high recovery, yield, and viability across a multitude of cell types with up to 99% purity within sorted populations. It can sort out of a large array of tube sizes which allows us to sort through large volumes of sample with up to 4 way simultaneous cell sorting to adapt to applications. It can sort into 8-well strips, slides, and plates for both single cell sorting and bulk populations of a cell type which can be cultured after through sterile sorting. It can analyse different light orientations to accommodate unique and niche applications that demand features lacking on other standard flow cytometers. For your next sort session, please make your booking on Mo-Flo XDP.

Flow cytometry sheath fluid

We have nine flow cytometers in the facility, which made use of three different type of sheath fluids.  Sheath fluid is the medium required for focusing the cell suspension into a single stream to be interrogated by the laser though a process known as hydrodynamic focusing.  On the analysers your sample will not come into contact with this fluid until after analysis and on the sorters they will combine with your sample after the laser.  The sorters use autoclaved PBS made by the media team on site, the Cytoflex uses sterile ddH2O, and the BD analysers use the proprietary sheath fluid, BD FACS Flow. PBS, BD FACS Flow and ddH2O can be used interchangeably on analysers with very little effect on data quality.

We are currently investigating moving away from BD FACS Flow for the BD analysers to either PBS or ddH2O.  We want to do this for a number of reasons; shipping 25kg boxes of liquid across countries is not good for the environment, the heavy boxes have been causing health and safety incidents (bad backs), and the benefit of using proprietary sheath is negligible to our users.  Over the next couple of months we will be testing alternatives on our own samples and will let you know the results.  Please contact us if you have any questions or concerns.

Cytokine analysis on flow cytometers

Completing multiple ELISAs can time consuming and can use up large volumes of precious sample.  To get around this you can multiplex your analysis using beads.  The basic idea is that you have a range of barcoded beads specific for each analyte and you do a sandwich ELISA on the beads.  The benefits are reduced sample volume and greater consistency, because you are using the same sample for all of the analytes.  This can be done on a flow cytometer using custom or premade kits:

BD: CBA

Information: https://www.bdj.co.jp/pdf/55-04_04-7900030-3-A1.pdf

Products: https://www.bdbiosciences.com/us/applications/research/bead-based-immunoassays/cba-kits/c/745475

Biolegend: LEGENDplex

Information and products: https://www.biolegend.com/legendplex

ELISAGenie: GeniePlex

Information and products: https://www.elisagenie.com/genieplex

Please contact us for advice if needed.

 

Application of the month: Apoptosis measurement

This is a method of distinguishing self-induced cell death from other causes of necrosis. Using Annexin V and PI provides the opportunity for researchers to investigate and comparably measure the difference in these different pathways for cellular death which can prove useful later for downstream applications and data analysis.

Using a live/dead stain in your experiment is essential to exclude cells that may exhibit unusual staining and are not the cells you want to analyse.  These stains only detect dead cells, not cells that have begun the cascade towards cell death.  This is why checking for apoptosis is useful prior to sorting or data analysis, as these cells may adversely affect your cell culture (release of cytokines, DAMPs, etc.) or display unusual staining patterns.

Phosphatidylserine is a phospholipid and component of the plasma membrane located on the inner leaflet of the cell. During apoptosis the plasma membrane is depolarised which inverts phosphatidylserine to the outer leaflet as a signal for the cell to be phagocytised. Annexin V is a cellular protein that binds to phosphatidylserine which can be conjugated to a fluorophore to determine the level of apoptosis within a sample population. Used in tandem with a simple live/dead stain such as Propidium Iodide, which binds to the DNA of cells which have compromised and permeable membranes, creates an assay to measure and compare levels of apoptosis and necrosis.

Mid Anglia Cytometry Club Meeting – July 17th at Sanger

We are delighted to be hosting the next Mid Anglia Cytometry Club (MACC) meeting on Wednesday 17th July at 1800 – late.


The program is:

1800-1845 – Light refreshments and introductions

1900-2030 – Scientific presentations:

Bee Ling Ng: Analysis of chromosomes on conventional flow cytometers

Annie Speak: High-throughput phenotyping to uncover novel immune regulating genes

A few short presentations on Cyto 2019

A word from our sponser: AntibodyGenie

2030 – Late – General chat and light refreshments

Presenters
Bee Ling Ng, Annie Speak

The location is the Wellcome Sanger Institute, EBI South building, Kendrew Lecture Theatre.

https://www.sanger.ac.uk/about/who-we-are/sanger-institute/find-us

If you would like to attend please contact Chris using ch15@sanger.ac.uk

There will be directions to the room at reception.


The following meeting will be held at the Cambridge Institute for Medical Research (CIMR) on 25th September 2019

Cellular Operations Open Day: Thur 29 Nov 1-4pm

The Cell Ops open day is an opportunity for you to discover the great in-house services available to researchers on campus. There will be tours, games and a chance to discuss your projects and how we can help you to complete them.

Come and meet the teams on Thursday 29th November 1pm-4pm in the Sulston building.  We will have someone at Sulston reception to let you in, no need to worry about access.

CGaP: E360

Cytometry: F234

High-throughput gene editing: F334

Bespoke gene editing: F334


About Us

CGaP

The Cellular Generation and Phenotyping Core Facility is a large cell biology core facility consisting of about 32 scientists from a variety of scientific backgrounds.  We work closely with faculty to carry out large scale cell biology projects to help further the institutes scientific aims.   We work across all areas of cell biology with particular expertise in stem cells, organoid production and genome wide CRISPR screening.

Cytometry

The Cytometry Core Facility is a dedicated scientific service offering investigators state of art instrumentation and expertise in flow cytometry.

  • Researcher operated cell analysers,
  • Staff and researcher operated cell sorters
  • Experimental design and consultation
  • Training on cell analysers, cell sorters and in data analysis

High-throughput Gene Editing

We have established highly efficient methods that take advantage of CRISPR-Cas9 technology to generate constitutive knockouts in iPS cells at scale. The scope of our service to faculty includes the provision of experience, advice and assistance in the design and implementation of routine gene editing projects in iPS cells.

Bespoke Gene Editing

We use CRISPR/Cas9 techniques to generate iPS cell lines involving more complex alleles.

  • Single nucleotide polymorphisms (SNPs) e.g. to replicate or repair disease alleles
  • Conditional alleles e.g. Cre-dependent FLIP alleles, chemically or optically induced degrons
  • Tagged alleles e.g. fluorescent reporters, affinity tags, degrons
  • Multiple compound mutations e.g. sets of mutations in a gene family

We look forward to meeting you on the 29th November

October 2018: Cytometry Advanced Workshop, Cell Ops open day, RA career day, Sam’s is RA champion, Jennie’s is QA Champion

September Newsletter: scholarships, meetings, lasers, cell counting, and the results from our annual satisfaction survey

The autumn equinox is approaching (23rd Sept) and so is the traditional Harvest Festival where we give thanks to all the great science we have been part of during the past year. The list of publications using the facility can be found here. We know there are more, because you are brilliant, so please fill in the gaps by telling us about yours.

This month we will be discussing scholarships, meetings, lasers, cell counting, and the results from our annual satisfaction survey.


Marylou Ingram Scholar Program

Marylou Ingram Scholars

The ISAC Marylou Ingram Scholars Program is a five-year career development program designed to enhance the scientific and leadership experiences of research leaders in the field of cytometry.
It provides leadership training, mentorship, presentation opportunities, membership to the Society, a subscription to Cytometry Part A, complimentary registration and partial travel funds to attend the annual CYTO conferences, and other valuable professional development activities.
Applications to the 2019-2023 program are being accepted until 30th October 2018.
Go to the ISAC website for more information.


News Bites

FT2 screen trial: For the next month, we will trial having the FT2 screens in portrait orientation. Let us know what you think using the feedback form on the bench.

MACC meeting: There will be a regional cytometry meeting at 6pm on Tuesday 18th Sept at CIMR. See the MACC website for details. Everyone is welcome (if you analyse single cells).

FCS Express: The trial of FCSExpress is ongoing and it has been well received. Email us if you would like to use the software for yourself. See FCSExpress’ features on YouTube.

RA day: There will be a research assistant career day held at Sanger on Thursday 13th Sept. Work out where you want to be in 5 years and how to get there. See Helix for more details (GRL only).


Coherent Connection

What do you do if you cannot visualise both the positive and negative populations on the same plot? You might need to titrate your antibodies or reporter protein. If this is not possible you could reduce the laser power of the instrument. On both Fortessa’s we have software called Coherent Connection installed which allow this. If you think you may need to do this let us know and we will show you how.
BE WARNED that if you do use it you MUST restore the laser power to the default (full power) settings after your experiment.


Volumetric Cell Counting

The Cytoflex has an awesome feature: volumetric cell counting. The Cytoflex aspirates sample using a peristaltic pump which means that it knows exactly what volume it has taken. This is recorded in the fcs file meaning that not only does CytExpert tell you the cell concentration, but you can work it out yourself using simple maths: event count divided by volume ($VOL keyword).
Magic!
It is much better then using counting beads on the other cytometers or looking down a microscope and it is more flexible then dedicated counters as you can work out the concentration of specific cell types using fluorescent antibodies.


Survey Results

In March we asked for your opinion of the facility and today you can see the results. The results are found in a Tableau Story (GRL only)
The headline figures are that 38 of you responded of which 74% said we provide a 5 out of 5 service. You find our new calendar system easy to use and if you could have more sort capacity, it would be in the evening.
We have read all the comments and have enacted many of them already. You can see the service status of the instruments on Confluence, we have advertised our phone numbers in the lab and we are planning an advanced flow cytometry course for users during the autumn.
If you disagree or have suggestions please let us know.

p.s. 76% of users find the newsletter interesting 😀


Planned Downtime and Past Incidents

Every Monday 10am-11am, all staff sorters, staff meeting
Every Tue morning both Sony’s are cleaned
4-5th Sept all day, One staff sorter, training course
10-11th Sept all day, One staff sorter, training course
12th Sept morning, all staff sorters, Cell Ops meeting
21st Sept, two staff sorters, staff training

Fortessa 2 went down one evening due to a power failure. A device was put into a socket that tripped the electric circuit. Unfortunately the UPS for FT2 was out of action at the time. All the other analysers are protected by a UPS. The UPS is due to be repaired and an engineer visited to reset the circuit.
Both Sony’s went down this month with faulty waste aspirators. They were fixed by the Sony engineer and Sony replaced the waste pump on Sony 1.



Flow Cytometry: The original single cell technology

August 2018 Newsletter: newly released instruments, our busy summer, FCSExpress, replaceable fluidics, exciting papers and planned down time

We finally had a little bit of rain to break up the boring monotony of sunshine at Sanger. This month we talk about newly released instruments, our busy summer, FCSExpress, replaceable fluidics, exciting papers and planned down time.

p.s. if you like the look of any of the instruments, or think that we should provide any other service, please let us know.


New instrumentation

Merck CellStream (released)
A 7 laser, 22 parameter benchtop flow cytometer using cameras to capture low-resolution cell images and transform these into intensity data with high fluorescence sensitivity.
Sony MA900 (released)
Sony’s new cell sorter and a direct upgrade of the SH800; 12 fluorescent parameters, 4 way sorting, and spatial separation between 2 sets of laser lines.
CyTek Auora (released)
The CyTek spectral flow cytometer has 3 laser lines and up to 50 detection parameters. It detects fluorophores using their entire spectrum allowing the separation of proteins normally not possible on traditional flow cytometers such as GFP and YFP.
Zellkraftwerk (released)
A high throughput chip cytometer that sequentially stains, images and washes using the same 5 fluorophores allowing quick automated cell imaging. It also ‘virtual sorts’.
BD Imaging Cytometer (soon, maybe)
Using technology from Omega Biosystems, an imaging flow cytometer that uses radio frequencies to image cells as well as analysing standard fluorescent information. *Note: we were not invited to the BD Cyto2018 event that we assume showed this
Ghost Cytometer (future)
Another imaging cytometer. The interesting thing about this one is that it also sorts. It uses the same technology as ghost imaging, just backwards!


Busy Bees

Cyto: Chris and Sam went to Cyto, the annual congress for cytometrists. We presented a poster on the work we have been doing to automate the lab and Chris gave a presentation on the Human Cell Atlas with colleagues from Uni of Sydney and The Garvan Institute of Medical Research.

CTLS: Bee, with Alex and Rachael, went to the Core Technologies for Life Sciences congress to represent Cell Ops. They went to share their experience and to learn how others run their own core facilities to help improve Sangers.

 

flowcytometryUK: Is the biannual conference of UK based cytometrists. Bee and Jennie went with a poster on spillover spread (ask Jennie, it is useful for designing flow panels). Here Bee is with our neighbours Maria Daly (LMB) and Esther Perez (Phenotyping Hub).

 

WTAC: We have had 30 students though the lab in the past week. First was the Wellcome Advanced Course on Single Cell Analysis, then there were sixth form students from around the UK doing a weeks work experience with the Malaria team.

 


FCSExpress

We have a 3 month trial license of FCSExpress. If you would like to use it please email us and you can have it on your PC or Mac. FCSExpress is an alternative to FlowJo and offers a completely different workflow. So if you can’t get along with FlowJo please give FCSExpress a try.


Replaceable Fluidics

We keep our instruments clean and the sorters as sterile as possible. We test this every two weeks and the sorters have not shown signs of contamination in 2018. However if you are still concerned about sterility or possible carry over of cellular debris then your best option is to use the Sony sorters as both the sort chip and sample line can be replaced easily. Talk to us for more details.


Papers

You’ve probably already heard about the Nature Biotechnology paper by our users Michael and Kärt about mutagenesis caused by CRISPR-Cas9 and it’s possible implications.
Also this month was a great paper in Cell Reports, with a fantastic gene map, by our users Meng, Jason, Kasia, Swee, Hiroko and Kosuke using a CRISPR knockout screen they discover naive pluripotency regulators in mouse ESCs.

We are very grateful to both teams for acknowledging the facility in their papers. These acknowledgments allow us to prove our utility to the institute and allows us to serve you with well maintained, reliable and state of the art instrumentation. Thank you.


Planned Downtime and Past Incidents

Every Monday 10am-11am, all staff sorters, staff meeting
Every Tue morning both Sony’s are cleaned
6th Aug all day, Influx, engineer visit
7th Aug morning, all instruments, cleaning
27th Aug, no staff, bank holiday

The Influx had become difficult to align. This was discovered to be that the laser shielding ring had become wedged between the nozzle assembly and the rest of the instrument. This was fixed and resulted in only 30 minutes of down time.



Flow Cytometry: The original single cell technology