Expand each of the subsections below for further information
General Facility Guidelines
- Keep the space clean and tidy.
- Return pipettes, pipette tips and other equipment after use.
- Ask for assistance if unsure.
- Report issues with cytometers.
- Perform the full cytometer cleaning procedure at the end of each session.
- Change sheath fluid and waste tanks as required.
- Have good sample prep, and filter samples to prevent blockages.
- Not overrun into the next user’s booking without asking next user for permission.
For immediate response during a session, users can contact facility staff via Instant messenger.
- Microsoft Teams (Flow Facility Issues)
The facility is open to all Imperial College staff and students, and to users from outside the College (subject to availability).
Usage of any instrument in the facility is only allowed after completion of training and induction with facility staff member (see the Training section).
All new users need to:
- register on PPMS,
- agree to the Data Protection Acknowledgment for administrative and finance purposes (in PPMS),
- create a financial account in ‘Profile’ > ‘New account authorization request’. Bookings will be rejected until a valid financial account is set up.
- request to join the facility biosafety database. An entry is required for each system. For unfixed bacteria, HG2 and class 2 GMOs samples, upload of an approved bio1 form will be required. Bio1 form information to include
Induction, card access, training and cell sorting will then be arranged with the facility staff. Users of Melody (522-SAFB), NanoFCM (522-SAFB), Fortessa II (638-SAFB), Calibur (3.22-Flowers) or CellStream (5.22-Flowers) require an additional induction to ensure safe CL2 working.
Read the Health and Safety section before using the facility for further information.
For especific information on Training procedures, please visit our 'Training' section.
To keep usage charges affordable, guarantee the survival of the facility and maximise access, it is essential that users are cooperative and disciplined with bookings.
- Every usage must be booked in advance in PPMS.
- PPMS tracking software will track user log in on cytometer computers. Please log out at the end of your session.
- The booking time must correspond to the actual usage time.
- Stop running samples 10-15 mins before the end of the booking to allow time to run the full cleaning procedure.
- Do not overrun into the next user´s booking without contacting the next user for permission.
- If usage is shorter, ‘Cancel rest of session’ in PPMS. Otherwise users will be charged 50% of the remaining time.
- Users can be notified if other users finish early by selecting ´Receive a notification by email if someone cancels a booking´ in PPMS.
- Less than 2 hours prior to the start time will be charged 50% of the time booked unless other user books that time slot.
- If over 30 min late for the session, users will still be charged for the time booked and recorded as a ‘no-show’.
- Within 24 hours prior to the start of sorting on Aria III will be charged 50% of the time booked.
Please contact facility staff if exceptional circumstances warrant the waiving of late cancellation fee.
Last users of the day cancelling a session are responsible to make sure that the instrument is switched off. They need to email the previous user, the facility staff or switch it off themselves.
Assistance should be requested at least 48hr prior to the booking to ensure staff availability. The facility staff will confirm availability, or will suggest alternative times if no assistance is available.
If changes to filter configurations are required, they can be requested in PPMS by ticking ‘Special Requirements form’ underneath the calendar before booking.
Requesting a sort
New users should follow these steps to request sorting:
- Create an account on PPMS FLOW_SAFB
- Once your account is approved, create a financial account (orders won’t be received until a valid financial account is set up). Click ‘Profile’ - ‘New account authorization request’
- Request to join the Facility Biosafety Database. Upload an approved Bio1 form and SOP if you intend to analyse unfixed CONTAINMENT LEVEL 2 (CL2) SAMPLES (HG2 pathogens. GM Class II organisms, human cells).
- Create a sort order for CL1_Aria or CL2_Aria depending on the Containment level of your sample. Melody sortings do not require a sort order.
Information to provide in sort order
Users will provide information on:
- Fluorochromes/Fluorescent Proteins being used,
- Cell type,
- Gating and sorting strategy. If you have performed this experiment before, write the name of the archive from your last sorting.
- Approximate number of cells in the sample (if known),
- Number and percentage of positive cells to be sorted (if known),
- Type of cell collection – number of populations to sort, bulk or single-cell sorting
- Type of collection device – 1.5mL Eppendorfs, 5mL tubes, 15mL tubes, cell culture plates or slides,
- Does the sample need to be chilled or heated?
- Is purity check required?
- Is a long pre-sort clean required? Recommended if culturing cells post-sorting or extracting RNA.
- Other - RNase provided to clean sort chamber, clean sample line with 96% EtOH, etc.
Items to bring to your session
Please be on time for your booking. If you are over 30 min late for the session, you will still be charged for the time booked.
- Filtered sample in Sort Preparation Buffer at a concentration of up to 5 million per mL - in a tube. Minimum volume 0.5mL
- Unstained sample. Minimum volume 0.2mL.
- Single colour controls for each fluorophore used (approx. half a million). Minimum volume 0.2mL.
- Other controls i.e. Isotypes, FMOs, live/dead cell stains. Minimum volume 0.2mL.
- Collection tube/plate/slide/dish with or without Sort Collection Buffer added.
- Extra Sort Preparation Buffer to dilute samples if needed.
- Extra Sort Collection Buffer.
|Unstained/untrasfected||This control should have gone through the same procedure as your other samples but without the addition of fluorophore.|
|Compensation (single colour) controls for each fluorophore||
For setting compensation between conflicting fluorophores.
Single-colour beads or cells (negative and positive in the same tube if possible).These controls need to be at least as bright as your brightest sample.
Fluorescence Minus One (FMO) controls for each fluorophore (recommended)
For setting gates through the absence of one fluorophore.
Beads or cells with all fluorophores except one added.
|Positive control (recommended)||If antibody is new, can use for example a cell line expressing that marker.|
|These controls are optional and should only be considered as an indication of non-specific binding. They need to be matched to the host species and isotype of your primary antibody.
They should be used at the same concentration as your antibody.
|If you do not have the proper controls for your sort, we will highlight there is no guarantee regarding the outcome of the sort, during your session.|
What are 5mL Falcon tubes and how to purchase them?
5ml Falcon Tubes are made by Beckton Dickinson BD:
- Sterile tubes with lids, Cat. Num. 352054.
- Non-sterile tubes (without lids), Cat. Num. 340345.
- 35µm Filter top tubes, Cat. Num. 352235.
Filtration and dislodging of samples can also be done with cell strainers less than 70µm.
|Up to 4-population simultaneous sorting||Up to 2-population simultaneous sorting||Single cell sorting||Single cell sorting|
|5mL Falcon||15mL Falcon||6 wells||Standard|
|1.5mL Eppendorf||24 wells||Frosted end|
|96 wells (incl. PCR)|
Sort Preparation Buffer
A good sort buffer is as follows:
- 1 X PBS
- 1mM EDTA or 10U/mL DNAse
- 25mM Hepes pH7.0
- 1% FBS (heat inactivated)
Cells can also be brought in 1 X PBS or media but only up to 2% FCS/Serum and no growth factors to avoid clumping of cells.
Sort Collection Buffer
Any buffer is allowed for sorting into, except Trizol due to aerosol danger. Please be aware of the dilution factor – particularly when it comes to reagents such as RNA later. Discuss your concerns with the facility staff.
Time to sort
This depends on the quality and concentration of the sample as well as the size of the cells within. Once the sample is running and the flow rate to get a good sorting efficiency is observed, an approximate time will be given.
Please see below for time required to sort a certain population percentage.
|Required number||Desired particles as % of total particles||Desired particles as % of total particles||Desired particles as % of total particles||Desired particles as % of total particles|
|103||8.3 min||50 sec||10 sec||1 sec|
|104||1.4 hr||8.3 min||1.7 min||10 sec|
|105||14 hr||1.4 hr||17 min||1.7 min|
|106||5.8 d||14 hr||2.8 hr||17 min|
|107||1.9 mo||5.8 d||1.2 d||2.8 hr|
|108||1.6 yr||1.9 mo||12 d||1.2 d|
Data and network
Network policy & virus protection
To avoid data loss and down times of the flow cytometers, users are required to stick strictly to the following policy:
- No USB sticks or storage devices of any kind can be inserted into any flow cytometer computer.
- Users are not allowed to install any software on any computers.
- Do not download files, even from trusted websites.
- Only allow scripts specifically from trusted websites, and only if needed for the visibility of the website.
- DO NOT ALLOW SCRIPTS GLOBALLY!
- Do not allow scripts for unknown websites or advertising pages.
At acquisition, all data are stored on local flow cytometers hard drives and should be backed up to Imperial H:drive or OneDrive.
- Databases will be backed-up weekly.
- To maintain databases, data will be deleted after one month.
- Deleted FCS files will be backed-up to an external hard drive.
- Users are responsible for storage and backup of their own data.
- Users are advised to export and save FCS files to their Drive or OneDrive immediately after acquisition to avoid data loss.
- To reuse experimental settings, users should export the experiment template.
Health and Safety
- All new users must complete a facility induction. This will be part of initial training.
- All users of Melody (522-SAFB), NanoFCM (522-SAFB), Fortessa II (638-SAFB), Calibur (3.22-Flowers) or CellStream (5.22-Flowers) will require additional induction for CL2 safe working.
User H&S records
Users are responsible to keep their Health and Safety records up to date.
- All users should log flow cytometry training in their personal competency records (FoNS, users from other faculties should contact their departmental Health and Safety team for information of training record system).
- All user projects should be logged with facility biosafety database, and bio1 RADAR for CL2 samples.
- Users should also read current facility and system-specific COP available on PPMS.
Prior to using any of the facility instruments, a risk assessment form needs to be completed. Please also refer to the Biosafety webpage for more information.
- A new entry in the facility biosafety database is required if new work is to be undertaken or changed. For unfixed bacteria, HG2 and class II organisms/human cells, upload of an approved bio1 form will be required. Bio1 form information to include
- All samples brought into the facility should be contained within lidded tubes and a secondary container i.e. a polystyrene box.
- Risk assessments must also be completed for use of hazardous chemicals.
- No eating or drinking in any flow facility labs.
- Tubes should be visually inspected for cracks before placing on cytometer, particularly in CL2 rooms and when working with pathogens and GMOs. If the tube is cracked, aerosols will be released when the system becomes pressurised.
- Sharps should be disposed of in yellow sharps bins.
- Glass should be disposed of in the glass bin.
- CL1 samples can be disposed of in the orange waste bins.
- CL2 samples must be returned to the user’s lab for disposal in accordance with local rules.
Normal working hours are defined as 07:00 – 18:00, Monday-Friday. For access outside these hours and days e.g. weekends and bank holidays, the user has to seek authorisation via the Faculty of Natural Sciences Lone Working Policy.
Staff/students using the SAFB Flow Cytometry Facility need to submit applications using the electronic system on the "Lone Working" Health & Safety webpages. Masters and BSc students and visitors without College status are not permitted to work at those times. Where workers from different faculties from Imperial College London are working in the facility, responsibility for the safety of those workers is shared between the worker’s own faculty and the Faculty of Natural Sciences.
Lasers in the cytometers are classed as IIIb – damage to the eye or skin can happen through direct beam viewing or specular reflections. However, lasers are enclosed during normal operation, therefore cytometers are considered Class I.
- Only an engineer or the flow cytometry staff can access the lasers.
- Exposed lasers need to be reported to the facility manager immediately.
The Imperial College course “Introduction to Laser Safety” is recommended but not compulsory for users.
Flow cytometers and their components operate at mains voltage. Live electrics are unexposed under normal operation, therefore risks are similar to those encountered in the use of other lab equipment.
- Modification/maintenance of the equipment is restricted to the facility manager.
- Only carbon dioxide fire extinguishers should be used around electrical equipment (including computers).
- Creation of fine mist of EtOH should be provided by using dripper bottles for disinfection with 70% EtOH.
Incident and near misses
Incidents and near misses should be reported to the facility manager and logged via SALUS.
If the fire alarm sounds:
- Users and facility staff should exit the building via the nearest fire exit.
- When in SAFB, stairs should be followed to basement and building exited at the back. The building should not be exited by crossing the SAFB lobby.
- The fire exit route should be followed round the back of Flowers building.
- The meeting point is in front of the Queen’s Tower.
If a fire is discovered in the facility:
- The nearest fire alarm should be activated.
- Fire extinguishers, located in the corridor outside the facility should only be operated by trained users, and if the blaze can be tackled safely.
- Faulty fire alarms should be reported immediately via SALUS.
Medical and security emergency
In the event of a medical emergency, security should be called on 4444. 999 should NOT be called.
Unauthorised access to the facility should be reported to the facility manager.
Acknowledging SAFB Flow Cytometry Facility in publications
The SAFB Flow Cytometry Facility is funded from a variety of sources to which we are accountable. It is very important for funding purposes to show that the Facility is well used. This means that all users are required to acknowledge SAFB Flow Cytometry Facility usage in their presentations or publications (please also email us a pdf of published manuscripts). Failure to do so will mean revocation of the permission to make bookings until further notice.
An example sentence would be:
“The Sir Alexander Fleming Building Flow Cytometry Facility at Imperial College London is part-supported by funding from …”
We also ask that you consider how much use you have made of the facility staff time. If this goes beyond basic training and support please consider whether to acknowledge the staff member or even include them as a co-author as appropriate.
Please use Report a Publication in the PPMS homepage as soon as possible – it is very easy and a big help.