FAQs
What do I need to do before using the WIMM Flow Cytometry Facility for the first time?
How far in advance is it necessary to book cell sorting?
What types of tubes and plates can be used for cell sorting?
How many different populations of cells can be sorted at the same time?
How many parameters/colours can be used in an experiment?
How do I know what fluorochromes can be used on a particular instrument?
What do I need to bring to the Facility?
What is the ideal concentration for samples?
How long will it take to sort/assay a sample?
How efficient is cell sorting?
What is the maximum number of colours that can be run in an experiment?
Answers
What do I need to do before using the WIMM Flow Cytometry Facility for the first time?
It is necessary to prepare a biosafety risk assessment for the samples that will be processed. This considers the potential risks associated with the samples, including, for example, does the sample contain pathogens or other potentially harmful agents? If you will be self-operating instruments it will also be necessary to complete a short-lab induction.
The lab is open 24 hours a day for internal staff and collaborators, visitors can use the lab between 9am and 5pm (Mon-Fri). Cell sorters normally operate from 10 am to 5.30 pm (Mon-Fri).
How far in advance is it necessary to book cell sorting?
Normally 1-2 weeks but it is often possible to get short-notice bookings
Trained operators can book self-assisted slots on all of the cell analysers using an online booking system. Bookings may be cancelled up to 8 hours before the start of the booking, cancellations with less notice will be chargeable.
Cell sorters can only be booked via the Flow Facility Team using a sort request form (https://forms.gle/67hMbU2hYFqqmcok9). It is advisable to book about 14 days in advance but it is often possible to get short-notice bookings. Bookings may be cancelled up to 24 hours before the start of the booking, cancellations with less notice will be chargeable.
What types of tubes and plates can be used for cell sorting?
1.5ml Eppendorf tubes, 12x75mm tubes and 15ml conical centrifuge tubes are suitable as sample containers. Cells can be sorted into similar tubes and in addition 0.5ml Eppendorf tubes. Cells can also be sorted into microplates and slides - a wide range of different plates can be used including 96 well and 384 well plates. Temperature control is available for both the sample tube and the collection tubes.
How many different populations of cells can be sorted at the same time?
All of our cell sorters can sort 4-ways but the BD S8 cell sorter can sort up to 6-ways. Cell sorting into plates is currently limited to one-way sorting, although it is possible to programme the sorter to sort different populations into a well/plate.
How many parameters/colours can be used in an experiment?
This varies depending on the instrument. The Sony MA900 4-laser cell sorter supports a maximum of 12 colours, in contrast, the BD S8 cell sorter can assay 30-50 parameters. All of the Facility’s sorters and analysers are equipped with at least 4 lasers (405nm; 488nm; 561nm; 640nm) and some have an additional 5th laser for UV excited fluorochromes
How do I know what fluorochromes can be used on a particular instrument?
Each instrument has an optical configuration table and examples of common fluorochromes that are compatible with each channel are listed. Please see the instruments pages. The tables do not contain all of the compatible fluorochromes so please ask the Facility staff for advice.
What do I need to bring to the Facility?
The Facility does not provide sample tubes or collection tubes and cell culture media. It does provide tubes for instrument QC and cleaning. So, in general, you should bring samples dispensed into suitable tubes (or plates if the autosamplers will be used) and also bring along collection tubes or plates for cell sorter collection. We provide gloves and cleaning materials and a small number of generic safety glasses. We recommend that you bring your personal lab coat and safety eyewear. It is compulsory to wear a lab coat, safety eyewear and gloves when operating flow cytometers.
What is the ideal concentration for samples?
In general, samples should be at a concentration of 1 to 10 million per ml. If for any reason this is too concentrated for certain applications please bring along some media to dilute the samples a little before acquisition. If samples are very dilute it may take an unusually long time to acquire/sort the samples. Please bear in mind that it only takes a few seconds to dilute a sample but it can take several minutes to concentrate a sample by centrifugation if that is necessary.
How long will it take to sort /assay a sample?
This will vary, but providing the sample is sufficiently concentrated it is possible to achieve acquisition rates of at least 10,000 cells per second on the cell analysers and between 2,000-20,000 cells per second on the cell sorters (nozzle dependant). As an example, if a sample is run at 7500 cells per second on a cell sorter and the target population has a frequency of 10% it will take about 22 mins to sort 1 million target cells.
How efficient is cell sorting?
It varies and there are a lot of factors to consider. Each cell sorter has a maximum theoretical sort speed but if samples are processed very quickly at the maximum rate, sorting yield/recovery will be very low. However, it is still possible to sort fast and achieve an efficiency of 90%. The actual number of cells recovered may vary depending on the fragility of the cell and other factors.
What is the maximum number of colours that can be run in an experiment?
There are several important factors to consider. On a conventional cytometer like the Fortessa with 4 lasers, it is determined by the number of channels or parameters, which is 15. On the Symphony A5, it is 31. On a spectral cytometer, like the BD S8, there is no defined limit but the restricting factor will be the number of available compatible fluorochromes. Panels of up to 60 colours have been developed for spectral instruments and it is likely that up to 100 will be possible within the next 10 years.