Training and Support
Overview
Training
You only need to bring a notepad. We don’t need any cells to train you on the instrument. The first induction addresses the basic use of the instrument and software, rather than establishing your specific experiment.
Please complete the following form to register with the facility.
Support
Find support on using our instruments. If you're having problems, read our troubleshooting information. If you are still experiencing issues, contact us.
Analysis of flow cytometry data can be daunting especially if you're new to the technology. If you require help with analysis using any software we are available to assist. Just contact any member of the team and we can help you with your experiments.
Health and safety
It is essential that all users of the FCCF adhere to local and university-wide health and safety regulations. Staff will ask you to provide relevant risk assessment documentation when you register with us and ask you to read and complete a SOPRA.
Further information regarding health and safety can be found on our website
Download our health and safety documentation (PDF: 672 KB)
Useful links
Educational sites
- Introduction to Flow Cytometry
- Flow Cytometry- A Basic Introduction (MG Ormerod)
- Fluorescence tutorials
- Chromocyte
Societies
Cytometry Tools
Using our instruments
Here are a few guidelines for support using our instruments.
The points below give you tips for using our instruments. If you have any other questions, please contact us.
Setting up a new experiment
What you'll need to bring with you depends on how complex the experiment is. Simply speaking, you'll need:
- unstained cells
- single antibody stained cells
- single stained compensation beads (if your panel requires compensation)
- unstained/negative compensation beads
- fully stained 'experimental' cells
Number of events
The number of events you need to acquire and record depends on:
- how rare your population of interest is
- the amount of debris in your sample
- the number of of dead/dying cells in your sample
All events
Your cells will be recorded as 'All Events' as you leave the stopping gate if they are:
- rare cell populations
- large amounts of debris
- large amounts of dead or dying cells
To get an accurate number of cells of interest recorded, make sure to change your stopping gate to the population of interest.
Flow rates
There are low medium and high flow rates.
The higher the flow rate, the faster your cells will run through the instrument.
You need to take care. Running the instrument on higher flow rates increases the spread of your populations. This can mask subtle shifts in fluorescence otherwise visible on lower flow rates.
Best practice is to pick a flow rate and be consistent between all samples.
Data and experiment storage
At the start of a new calendar month all data is backed up. Experiments are removed from your user profile.
To find your experiments look in the R drive before you come to the instrument. Locate them by instrument configuration, year, month, experiments, and user name.
Alternatively a copy is held locally on each instrument's D: drive.
Cell Sorting
We have three cell sorters across our two sites. On these pages you'll find support for using them. You can find information about them on our equipment pages.
Cancellation policy
Sorts can be cancelled fully with no charge up to 24hrs before the start time.
Any sorts that are cancelled within 24hrs of starting but before 5pm the day before, are charged at 1/2cost with a 30 minute minimum time.
Sorts cancelled after 5pm the day before and within 24hrs are fully charged.
If someone can be found to fill a cancelled sort slot then no charges are levied against the initial user. All cancellation cases are reviewed by FCCF staff personally so please contact us to discuss any and all cancellations.
Bringing your cells
Number of cells to bring
The number of cells you need to bring depends on:
-
How many cells you require for downstream work.
-
How rare your cell population is within your pre-sorted sample.
If you know roughly these two variables you can make an estimate for how many cells you need to bring.
Calculating your cell estimate
We recommend that you bring around 20% more cells than your estimate. This is to allow for inaccuracies such as:
-
a percentage of any pre-sorted sample will be non-viable (not desirable for sorting)
-
any cell counting method has a level of inaccuracy
-
finding that you have fewer cells in your pre-sorted sample than you had thought
You should also decide whether you are counting your cells before or after staining and washing. Numbers will differ based upon when you count.
Our sorters all give a cell count of the sorted cells. This number is at best 85% accurate.
If your downstream application requires a minimum number of cells, please allow a 15-20% margin of error extra within the sorted population.
Example calculation
You want 100,000 total cells. Your target population is 1% of the total cell population.
You need:
100,000 x 100 = 10,000,000 cells + 20% extra = 12,000,000 total cells needed.
Buffer and concentration
We recommend that you bring your cells in FACS tubes in a buffer the cells are happiest in.
Decrease the protein concentration of the buffer to a maximum of 2%.
We recommend a cell concentration of no more than 2x107 per ml.
Bring an extra buffer to the sort as we may need to dilute your cell sample.
Filtering your cells
If your cells are adherent or there is a large amount of dead and dying cells in the population you may find that cell aggregates form (clumping).
This can easily block the sorter and delay your sort significantly.
To ensure this doesn’t happen we may ask you to filter your cells to remove aggregates.
We sell 30um filters at the facility. These are not sold sterile but you can also bring your own.
Rule of thumb is that the filter size should be around half that of the sort nozzle used.
For example, cells that are to be sorted through a 70 um nozzle = 30 um filter. Cells through a 100 um nozzle=50 um filter.
It is pointless to filter cells through a 100 um filter then expect them to pass freely though a nozzle of the same diameter.
Collecting your cells
Tubes, plates, microscopic slides
We have the capacity to sort into:
- tubes (eppendorfs, FACS Tubes, 15ml Falcons)
- plates (384 wells to 6 wells)
- microscope slides
Specify what you are planning to collect in before the sort is set up to avoid any delays.
The maximum number of simultaneous sorted populations are:
- Eppendorf -4 (6 on Astrios)
- FACS Tube -4 (6 on Astrios)
- 15ml Tube -2
- Microscope slide -3
- Any plate format -1
Buffer
We recommend that you collect your cells into a buffer. This avoids stressing the sorted sample.
The larger the volume of collection buffer the lower the stress on the cell. But, this will mean you can collect fewer cells per tube.
Collect your cells into neat FBS/FCS or high concentration FBS/FCS cellular media. This can improve the survival of your sorted sample.
Sorting process
Number of cells we can sort
The number of cells we can sort in an hour depends on the size of your cells and therefore the size of the sort nozzle we can use.
The smaller the sort nozzle the faster we can sort. 70um is the smallest nozzle. We then have 85, 100 and 130um nozzles.
| Fusion, Aria II and Aria III | Astrios | |
|---|---|---|
| 70um | 30 million/hour (60PSI) | 40-50 million/hour (60PSI) |
| 100um | 15 million/hour (20PSI) | 20-30 million/hour (30PSI) |
Nozzle choice
The nozzle choice is dependent upon the size of the cells in your sample.
As a rule the nozzle should be 4x bigger than the largest cells in your sample.
As a general rule:
- PBMCs (8-12um) require the 70 or 85um nozzle
- cell lines and bigger primary cells (12-20) require the 100um nozzle
- anything larger requires the 130um nozzle
Be aware that the smaller nozzles have higher pressures. Some smaller cells do not like this pressure. They can be sorted on larger nozzles with a resulting drop in rate.
Sterility process
Our sorters are all sampled for sterility every week. They're cleaned thoroughly on a regular basis.
We also shut our sorters down with 70% ethanol every night to avoid any contamination and to preserve the sterility.
Post-sort culturing is common practice and infections are rare but can occur.
If contamination occurs
If your sorted cells are infected, let us know. We will identify if it was the sorter or some other source of contamination.
To decrease the risk of contamination, prepare your sample in tissue culture facilities with proper aseptic technique where required.
Other equipment in the core facility (such as our lyse wash assistant) is not screened for contamination. This can be a source of contamination if used before sorting.
Temperature for sorting
We can adjust the temperature of your sorted sample in two locations:
- Your pre-sorted tube can be controlled at 4, 20, 37, and 42oC within the instrument.
- Your collection tubes/plates/slides can be controlled to any temperature between 5oC and 42oC.
The temperature you sort at is dependent on your cells and your downstream assay.
If you want to culture your cells after sorting it may be preferable to sort at 37oC.
For other protein or nucleic acid work, cooler temperatures may be preferable. This can preserve the integrity of your sample in the collection device.
If in doubt as always it is best to ask a member of the FCCF team.
Encountering problems
Here are a few points for support if you're having trouble using our instruments.
The points below will help you troubleshoot common issues. If you have any other questions, please contact us.
Trouble acquiring events
Remember to click the 'little green arrow' to the left hand side of your tube.If this is not clicked, the software will not let you acquire.
Cells have disappeared
If your cells have disappeared, check that the FSC and SSC settings the same as previously used.The cytometer may have reverted to higher FSC and SSC, pushing your cells off axis.
No events appearing
If no events appear, it is likely that the instrument is either blocked or has air bubbles causing problems inside the flow cell.Contact a member of staff to help you rectify the issue.Also check your sample doesn’t contain any clumps of cells which can block the instrument.If you can see any particulate material this will block the instrument. Your sample will require filtering before running again.
Software
Our software helps you analyse acquired data quickly and efficiently, remote from the instruments.
Accessing the software
You can access a comprehensive suite of flow cytometry analysis software at each of our two sites. If you require any help in using the software below to analyse your data please contact a member of the FCCF team and we will assist you with your analysis.
FCS Express 6
We have a site license for this software. Visit their website for information and Contact Us for access
Diva Software
This is for running BD instrumentation. Download the reference guide (PDF: 3.7 MB).
FCAP Array Software
This is for bead based multiplex assays (CBA assays).
FlowJo
This is for offline analysis of data. Visit their website for information and technical support.
Modfit Software
This is for curve fitting ie DNA analysis. Visit their website for information and technical support.
Consumables
Ordering consumables
We sell a variety of consumables for use in the FCCF such as:
- FACS Tubes (125 per pack)
- 35uM cell filters
- Tali analysis slides
- Seahorse media and plates
Please complete orders in the PPMS system to order consumables for collection.
Protocols
The FCCF has tried and tested protocols which can improve the quality of your data. Newcastle University staff and students can access these protocols here [Internal].