Flow Cytometry

@ University of Manchester

Dr Gareth Howell setting up a cell sort on the BD Influx Cell Sorter in the CTF building flow cytometry facility laboratory


Welcome to the pages of the flow cytometry core facility within the Faculty of Biology, Medicine and Health at the University of Manchester.

Our facility offers researchers access to state-of-the-art flow cytometry analysis and sorting equipment, high parameter mass cytometry and imaging cytometry. The facility is managed full time by an experienced Senior Experimental Officer and is supported by two technical level staff.

We have available 6 flow cytometer analysers and 2 cell sorters. Additionally, we have a Helios-CyTOF for suspension mass cytometry, Hyperion Imaging Mass Cytometer and an ImageStreamX. Details of these instruments can be found in the links above.

Please contact Dr Gareth Howell (Facility Manager) for further information relating to accessing the facility.

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Accessing the Facility

Register your account

In order to access the booking form and equipment calendar you will firstly have to register your details with the PPMS booking system

This can be accessed at http://tinyurl.com/bookflowcyt

You will be required to create a project after your registration has been accepted. You will then be able to request training, book the analysers or order services (cell sorts, submit samples to run on the CyTOF etc).

Analysis equipment

Firstly, contact the Facility Manager Dr Gareth Howell either by email (gareth.howell@manchester.ac.uk) or drop into his office in the CTF Building for a discussion about your project and what you hope to gain from using flow cytometry technology.

Training is then arranged one-to-one on the most appropriate system and is best performed with the researcher's samples as this will provide the most contextual training experience.

Once the above stages have been completed the user will be given access to the online booking system for the instrument(s) they were trained on and can access these systems whenever the require.

Detailed information about the equipment we have in the Facility can be accessed under the 'Equipment and Systems' page linked above.

Sorting equipment

Cell sorting is undertaken by trained facility staff (Dr Gareth Howell (CTF building) and Mr David Chapman (Michael Smith building).

Sorting appointments are arranged at a mutually convenient time following discussion with the sort operator.

Researchers will then be required to submit an 'Order' through the PPMS system which will then be accepted by the operator.

Following the sorting experiment the operator will edit the sort order and submit this to finance.

Details regarding sample preparation etc can be found on the cell sorting page

Data Analysis

Flowjo

We use Flowjo to perform third-party data analysis of the data produced in the Facility.

For more information and training resources on Flowjo please visit the website at http://flowjo.com

We have a number of Flowjo dongles that are available for loan from the Facility.

Please contact us for further information.

Workshops and Training

We run a series of in-house training workshops on all aspects of cytometry.

  • New User Training on Analysers

New users will be requited to attend a laboratory induction session where local rules and building health and safety are covered.

One-to-one training on operation of the analysers will be arranged s required and is detailed in the 'Accessing the Facility' section above.

  • TechnoShort workshops

These are a series of 3-hour long workshops that over consecutive weeks cover the following topics:

  • Introduction to flow cytometry

  • Multicolour panel design

  • Compensation in flow cytometry data

Classes are restricted to 8 attendees due to space in the lab

  • Imagestream workshop

We are planning to provide an introduction to the Imagestream technology in a workshop.

Please e-mail Gareth if you are interested, and updates will be posted here


Resources

Panel Design

Please contact the Facility to discuss any panel design questions you may have.

We also run a short workshop 'Multicolour panel design' as part of the Technoshort series of workshops (detailed above).

To aid in panel design please read below about published optimized panels (OMIPs)

Flurochrome Brightness Index

Not all flurochromes are the same and one of the challenges of designing a multicolor flow cytometry experiment is balancng the anticipated expression level of the antign with the relaive brightness of the flurochrome. To aid this there are 'Brightness Index' tables where the relative brightness of common flurochromes are given.

Click here for the brightness index table (opens a new tab)

OMIPs (Optimized Panels)

Optimized Multicolor Immunofluorescence Panel (OMIP) is a special peer-reviewed Cytometry Part A publication type that reports on newly designed and optimized multicolor panels for flow cytometry, fluorescence microscopy, image cytometry, and other polychromatic fluorescence-based methods. The first two OMIPs were published in the September 2010 issue of the Journal.

OMIPs are aimed: (1) to alleviate the development time for researchers in need of the same or highly similar panels, (2) to provide a starting point for the creation of novel OMIPs, and (3) to give the developers of the panels credit via citation or the publication.

An OMIP consists of two portions:

i. A two-page printed component includes basic information, such as the title, a summary table, a brief narrative, a reagent table, an example staining figure and a detailed comparison with similar OMIPs.

ii. An online component includes significant amount of technical details, such as panel development strategy, cross-references to related panels, exact staining protocol, instrument configuration and reagent information.

For more information on OMIPs, please refer to the following articles:

OMIPs—Orchestrating multiplexity in polychromatic science

Mario Roederer and Attila Tárnok

Cytometry, 77A: 811–812.

Publication of optimized multicolor immunofluorescence panels

Yolanda Mahnke, Pratip Chattopadhyay, and Mario Roederer

Cytometry, 77A: 814–818


Guidelines to use of the facility equipment

**NEW** Mandatory filtering of samples

In order to reduce the possibility of clogs and blockages, all samples, unless pre-arranged with the Facility manager, must be filtered (<80um) at the flow cytometer, even if samples were filtered earlier in the day at the lab bench.

The facility will not provide filters but can recommend a suitable solution.

Altered cleaning protocol (please allow 15 minutes at the end of your session to perform this cleaning procedure)

In Diva create a new tube within your experiment to record your cleaning samples

Set the events to record to one hundred million (100,000,000) and the Stopping time (sec) to 600 seconds

Place a ¾ full tube of 5% contrad on the sample port

Leave the arm open and run for 20 seconds

Close the arm and run on High for 10 minutes

Record this within Diva

Create a new tube in Diva and change the Stopping time (sec) to 300 seconds

Replace the tube with a tube of dH2O

Leave the arm open and run for 20 seconds

Close the arm and run on High for 5 minutes

Record this within Diva

Leave the water in place and put the fluidics on Standby or follow the shut down procedure as appropriate.

A remainder of other good practise

Data storage

Do not use FACSDiva for long-term storage of data. Please ensure you export data and delete old experiments from the FACSDiva software.

Data will be deleted without warning if it is over 1 month old from FACSDiva.

No USB or external data drives

There is a zero use policy of USB sticks or external data drives. Please do not use these on any of the machines within the facility (excludes FACSVerse).

To export data either email it to yourself (note there is limited internet access on computers connected to FACS machines in the facility) or use the flow data server for secure transfer of data. Please see PPMS – Documents for information on how to access this server.

Sheath and waste

Please check the level of the sheath fluid in the silver tank at the beginning of your session and ensure the waste in below the black line on the waste bottle. Fill or empty as appropriate.

At the end of your session fill up the sheath tank or empty the waste as appropriate.


Publication acknowledgement

Please remember to acknowledge the Core and our support grants in your papers if you accessed equipment, services, expertise, or data generated at the laboratory.

For detailed information on the funding source for the equipment click on the Equipment and systems page.

Suggested acknowledgement reference:

"We thanks the University of Manchester Flow Cytometry Core Facility for assistance with flow cytometry/ sorting/ discussions. The Flow Cytometry Core Facility is supported in part by the University of Manchester with assistance from <insert funding body>.