Reminder: Data is deleted off the flow cytometry analysers after 14 days.

A reminder that our current recommendation is to export your data onto your home directory (that is automatically mapped upon logging into the flow cytometry analysers) from DIVA once you have finished acquiring your samples. If you need to use a USB to offload your data, please copy the FCS files from the server, as this will ensure you will have a server copy of your data files that will be automatically backed up.

An updated data policy that will be adopted by the Scientific Platforms is currently under review. We will update users once this is implemented.

New display screen in the WRHFlow / Imaging Shared Lab

A new HD display screen has been installed in the shared lab. The screen is viewable from the level 2 café area and has been installed to highlight features of the Scientific Platforms as well as screen educational content. Screening will begin next week so keep a lookout for content!

HTS acquisition on the LSRII

The LSRII is equipped with a high throughput auto sampler that can be utilised to automate sample acquisition from 96 and 384 well plates. The HTS system minimizes carryover, provides user definable mixing and sample introduction protocols and is excellent for high number of samples. WRHFlow now has a supply of sterile 96 well V bottom plates and lids available for purchase through PPMS for high throughput sample staining, washing, and acquisition on the LSRII. Sterile V bottom plates with lids cost $6 and can be purchased via PPMS. Training can be requested through PPMS.

Christmas Closedown

Please note that WRHFlow will be closed from Christmas Eve, 24^th December 2017 until Monday 1^st January 2018 inclusive. There will no QC run on the flow cytometer analysers if there are bookings during this period. Please consider this and ensure a QC has been run before running your samples during this period if you need to use the instrument.

The sorters will not be operational on Friday the 22^nd of December as they will be shut down for the Christmas Closedown. The sorters will also not be open for bookings on Tuesday the 2^nd of January as they will be brought back online on this day. The first available day for a sort for researchers will be the 3^rd of January 2018.

There will be reduced facility support in place during this period and it is requested that only essential research work is undertaken. Any work during the Christmas closedown will need to be approved by your Research Supervisor/Manager in advance. If staff are required to work during this period, Research Supervisors/Managers are asked to send an email to Mark Shannon, Research Manager by 30 November 2017.

Repeat notice - Flow cytometer analysers baselines updating progressively in November 2017

From 2015 to the current, we have maintained the same target MFIs on the 2 CantoIIs and the LSRII via an almost daily quality control (note: Fortessa baseline had been updated 23/5/2017). This has allowed users to utilise application settings for longitudinal studies. The below notice is for users currently using application settings on the instruments.

As the original bead lot in use has expired we are required to establish new target MFIs (also known as baselines). This is also necessary when major changes have occurred on the instrument. Major notable changes that have occurred since 2015 include a new flow cell installation on both CantoII instruments, a new red laser on the LSRII and a new UV detector / blue laser on the Fortessa.

To those users using application settings and who wish to continue with existing target MFIs, know that you will need to save new application settings moving forward as we will record new baselines progressively in November 2017 as per the below schedule after the baseline has changed. This needs to be done once only.

The recommended method that would work for users who have not recorded beads in prior experiments, would be to switch to the existing baseline (the one that existing application settings were saved on), run a CS&T using the previous bead lot, make a new experiment, apply existing application settings, copy the voltages, switch back to the new baseline, run a CS&T using the new bead lot, make a new experiment, copy the voltages over to the new experiment, and save new application settings. Of course, this will cater for all FL detectors. If any help is required please contact WRHflow well in advance as a mistake would propagate to other users. The method is outlined below.

1) Run CS&T > Cytometer > CST > Performance check > using bead lot #64706 (old)

2) Exit CS&T, make a new experiment, apply your existing application settings.

3) Record your voltages!

4) Run CS&T > Cytometer > CST > Performance check > using bead lot #70644 (new)

5) Exit CS&T, make a new experiment, manually update your voltages.

6) Save new application settings with a distinctive name.

7) Your application settings will now be linked to the new bead lot number which we expect to last for ~ 1 year.

Ensure the above process is completed once started – to ensure the most recent CS&T report is from the #70644 bead lot, as the instrument uses the last CS&T data acquired.

New Baseline Implementation Schedule

Please remember we recommend compensation controls to be made and acquired in a similar manner and in parallel with your samples. We also recommend running and recording calibration beads both before and after your acquisitions to not only show the instrument was stable throughout the acquisition but to provide target MFIs in the event that application settings cannot be used on subsequent days.

Tip – Understanding negative fluorescence values and baseline restore

You may find yourself thinking it odd to have negative fluorescence values being reported in flow data files when the absence of fluorescence on a particle, one may think, should equate to either a zero or a low positive value. This can however occur because the values reported are arbitrary measurements of fluorescence on cells due to digitization.

When the fluorescence is detected off the PMT, the voltage measurement has ‘baseline’ subtracted from it. Baseline is measured during the time between events triggering on the instrument. Even when you are acquiring 20 000 cells a second there is more time when the lasers have no particles than particles passing through them. The instruments have error associated with the measurement of positive particles, negative particles and no particles (the baseline measurement). For ‘negative’ cells (cells with little or no fluorescence), there's a chance that the measurement for the cell will be less than the measurement for the baseline and therefore after subtraction of the baseline from the signal, the measurement can be less than zero. Additionally, compensation value estimations that are subtracted from channels when performing compensation can magnify these negative values significantly sometimes.

Negative values can be more pronounced if samples are not washed adequately and are acquired on a cytometer. As unbound fluorescently labelled antibodies that make it into the core stream will be result in a higher baseline that needs to be subtracted from event measurements. A high acquisition flow compared to a low acquisition flow will widen the core stream increasing the cytometer baseline value and reducing sensitivity (this is one reason why a narrow core stream is better for high parameter phenotyping). A high baseline and high errors can increasingly result in negative populations that may widen and or have extreme negative values. See below example.

The take home messages are

-washing your cells free of unbound antibody may improve the sensitivity of your panel

-negative values are normal in a digital instrument, extreme negative values however can indicate baseline restore problems

-adjusting the flow rate of your sample may influence the baseline as the core stream may widen/narrow changing the baseline value

2000 negative compensation beads acquired on high on a CantoII showing increased levels of unbound APC antibody can result in decreased fluorescence and increases in the rSD of the negative population due to increased background.

Orange = 1/1000 unbound APC antibody

Red = 1/250 unbound APC antibody

Blue = 1/100 unbound APC antibody

ARGUS Virtual PC – WRHFlow allocation – phase 1 pilot complete!

WRHFlow is participating in USYD’s Virtual Research Desktop (VRD) project. WRHFlow have a Medium VRD allocation (8 vCPU’s – Intel® Xeon® CPUs, 4 vGPU’s, 32 GB RAM and 300 GB SSD hard disk) with FlowJo 10.4,Matlab 2017a, Cell Profiler, Huygens and FijiImageJ installed on it. The instrument is bookable via PPMS and is ideal for long computing tasks executed with a GUI, i.e. FlowJo analysis. This VRD has the advantage of being powerful and accessible from anywhere with an internet connection (although we only recommend uploading data to it using the a wired LAN connection at WIMR or on Campus). Currently we have 1 seat available at a time, but in phase 2 we are looking at trialing multiple concurrent logins.

If you are interested in using the Argus platform please email wrh.flow@sydney.edu.au for instructions, booking and access terms.

More information can be found here https://rc.sydney.edu.au/argus-user-guide/getting-started.html

Visiting Scientist – Qiong Dong

WRHFlow has a visiting scientist, Qiong Dong, from Changsha Xiangya Hospital in Changsha, China. Qiong is visiting us on a scholarship to advance her knowledge in flow cytometry data generation, instrumentation and shared resource lab management. Currently she is based at The Third Xiangya Hospital of Central South University, and works on regulatory T cell isolation, porcine pancreas harvesting, islet isolation and mesenchymal stem cell isolation and culture. She will be with us from November 6^th 2017 until January 8th 2018. Please make her welcome if you see her in the flow labs.

Art in Science – competition closes today – 17/11/2017!

http://westmead.org.au/events/art-in-science-competition/art-in-science-submission

Remember to submit your entries (including flow cytometry analysis data) for an excellent chance of winning a prize!

Upcoming tutorial session run by WRHFlow – 4^th October 2017

WRHflow will be holding a tutorial session aimed at both beginner and advanced flow cytometry users on the 4^th of October 2017. The tutorials will be split into two sessions, one aimed towards new users, focusing on key concepts and setting up of the instrument, followed by an advanced session focusing on flow cytometry tips and tricks to ensure the generation of quality data and data analysis.

We will be holding 2 upcoming seminars on the 4^th of October 2017 @ 2pm & 3pm

2-3pm will be a basic introduction to flow cytometry for users who are thinking about using flow cytometry

3-4pm will be intended for current instrument users providing tips and tricks for generating quality data

Cell sorter contamination testing in place

The cell sorters undergo regular preventative maintenance that includes a 0.22um inline filter replacement in addition to sample and sheath line replacements. Various components of the instrument are also regularly autoclaved to avert downstream sorted sample contamination. In the past 3 years, seldom has WRHflow received enquiries regarding the sterility of the sorters due to sorted sample contamination, owing largely to the current practice employed in the facility, including the recommendation of antibiotics in collection tubes where possible.

An additional measure that we have now implemented consists of regular microbial and mycoplasma testing. The monthly testing consists of sampling the sheath tank, the ejected stream and the sample tube.

The current results were negative for mycoplasma in both the AriaIII and the Influx cell sorters, with <1CFU/mL in the ejected stream for the ARIAIII and the Influx. We are still in the process of establishing acceptable baselines for various samples and will continue to develop methods to ensure resulting sterile conditions for the cell sorters.

The testing has resulted in changes that include autoclavable wash bottles and sterile liquids to be used when SIP flushing as well as sheath tank handling changes to ensure aseptic conditions are maintained.

Tip - Viewing time on X axis and letting samples stabilize.

Please remember to visualise the time parameter when acquiring and analysing your data. This can help spot instrument issues, blockages and or the settling of cells, importantly, before you finish your sample. Always plot the last laser to be used along time as below.

Left: Instrument fluidics functioning well, sample stable after 2-3 seconds.

Right: Instrument fluidics functioning poorly, samples requiring >20seconds to stabilise

1M HEPES buffer in collection tubes – now available

We frequently receive questions regarding sort/collection tubes and what should be in them for cell sorting. I have prepared the below as a basic guide to frequently used sort/collection tubes buffers.

HEPES has been used in other flow laboratories, with success, to alleviate the pH changes that can occur in sort/collection tubes. PH changes can not only occur in the samples to be sorted (that can be kept at pressures upwards of 5-70psi for extended periods) but also in collection tubes. PBS/media containing carbonated based pH buffers are prone to pH change when not in a 5% CO2 environment such as an incubator.

We now have sterile 1M HEPES aliquots in the cell sorting labs that can be added to the sort/collection tubes upon request. As an example, to make a 20mM final concentration, 20uL needs to be added, per mL of sample, from WRHFlow 1M stocks. It is okay to have both bicarbonate and HEPES based buffering systems concurrently in samples.

Note: It is important that not only your cells, but your downstream application is compatible with your selected buffer protocol and that you consider how changing sort buffers by adding HEPES may impact previous/future data generation before implementing any changes. Some common application examples that can require specific buffers include annexin V staining, the calcium flux assay, nuclei sorting, single cell sorts and RNA/DNA extractions.

What can I bring my samples that is to be sorted in?

Vessel:

AriaIII cell sorter – accepts Eppendorf tubes, 5/15mL falcon/FACS tubes as inputs.

Influx cell sorter – accepts 5mL polypropylene FACS tubes as inputs.

Buffer:

Cells to be sorted in

(1-5% FCS/FBS OR 1-2.5% BSA)

in

(PBS Ca/Mg++ free OR HBSS OR media, preferred without pH indicator)

recommended with

(10-25mM HEPES)

with/out, by requirement

(1-5mM EDTA OR DNase + MgCl₂ OR custom buffer)

What to collect my samples into?

Vessel:

AriaIII or Influx – 96 well, 384 well, Eppendorf tubes, 5/15/50mL falcon tubes

Buffer:

Appropriate collection vessel coated with or containing

(100% FCS/FBS – with a final concentration after sorting cells of >10% OR cell culture media OR RNA/DNA extraction specific buffer OR custom buffer)

recommended with

(10-30mM HEPES – with a final concentration after sorting of cells >5mM & <30mM HEPES)

Flow cytometer analysers baselines updating progressively in November 2017

From 2015 to the current, we have maintained the same target MFIs on the 2 CantoIIs and the LSRII via an almost daily quality control (note: Fortessa baseline had been updated 23/5/2017). This has allowed users to utilise application settings for longitudinal studies. The below notice is for users currently using application settings on the instruments.

As the original bead lot in use has expired we are required to establish new target MFIs (also known as baselines). This is also necessary when major changes have occurred on the instrument. Major notable changes that have occurred since 2015 include a new flow cell installation on both CantoII instruments, a new red laser on the LSRII and a new UV detector / blue laser on the Fortessa.

To those users using application settings and who wish to continue with existing target MFIs, know that you will need to save new application settings moving forward as we will record new baselines progressively in November 2017 as per the below schedule.

One means to do this would be to match positive bead signal intensities of all FL detectors, with previously recorded data files, especially CS&T beads or another QC bead, on an experiment post the new baseline recording, followed by saving new application settings. This does mean looking at a statistic and matching the recorded signal with current signals.

Another method that would work for users who have not recorded beads in prior experiments, would be to switch to the existing baseline (the one that existing application settings were saved on), run a CS&T using the previous bead lot, make a new experiment, apply existing application settings, copy the voltages, switch back to the new baseline, run a CS&T using the new bead lot, make a new experiment, copy the voltages over to the new experiment, and save new application settings. Of course, this will cater for all FL detectors. If any help is required please contact WRHflow well in advance as a mistake would propagate to other users. The method is outlined below.

1) Run CS&T > Cytometer > CST > Performance check > using bead lot #64706 (old)

2) Exit CS&T, make a new experiment, apply your existing application settings.

3) Record your voltages!

4) Run CS&T > Cytometer > CST > Performance check > using bead lot #70644 (new)

5) Exit CS&T, make a new experiment, manually update your voltages.

6) Save new application settings with a distinctive name.

7) Your application settings will now be linked to the new bead lot number which we expect to last for ~ 1 year.

Ensure the above process is completed once started – to ensure the most recent CS&T report is from the #70644 bead lot, as the instrument uses the last CS&T data acquired.

New Baseline Implementation Schedule

Please remember we recommend compensation controls to be made and acquired in a similar manner and in parallel with your samples. We also recommend running and recording calibration beads both before and after your acquisitions to not only show the instrument was stable throughout the acquisition but to provide target MFIs in the event that application settings cannot be used on subsequent days.

Reagents available from WRHflow – Brilliant Stain Buffer

To assist researchers in panel design and experiment outcomes we have reagents available for immediate pickup via orders placed through PPMS. Items include: DAPI, BD Brilliant Stain Buffer, Human RBC lysing solution, FACS tubes with cell strainer caps, GentleMACS tubes, with other reagents to be added as they arrive.

WRHFlow 3D printer – now accessible!

3D printing allows rapid prototyping and manufacture of items useful in a research institute. The technology is quick to grasp and the resultant products are extremely beneficial. WRHFlow has acquired an Ultimaker 3+ to increase the capabilities of the flow and imaging facilities as well as advance medical research within the Hub.

This technology has been used to 3D print cell sorter collection blocks, stage inserts, live animal imaging mounts, and live animal handling tools. Others have used it to print magnetic tube racks, liquid handling robots, electron microscopy staining apparatus, educational aids showing cellular components or their research, as well as innumerable other uses.

The 3D printer is available to all researchers that are part of the Westmead Research Hub, enabling novel design and manufacture of objects to increase research quality outcomes. I urge and welcome all researchers to use the printer by contacting suat.dervish@sydney.edu.au

There is a considerable number of items already designed that are freely available on repositories such as:

https://3dprint.nih.gov/

https://www.thingiverse.com/

https://grabcad.com/

Instrument specifications include

• 20-200um Z resolution,
• 20-100^oC heat bed,
• print dimensions of 215x215x300mm
• active levelling,
• dual extrusion print heads,
• Materials currently available include PLA, PVA, PP, CPE, CPE+

Left: The Ultimaker 3+ extended installed in the flow cytometry analyser lab

Right: Ultimaker 3+ in action completing a test print

Monthly User Group Meetings

Thank you to those who attended the WRHflow first user group meeting. The meetings will be held on the first Wednesday of the Month from - 4.00pm-5.00pm in the Level 2 Conference Room C2.20 where flow cytometry topics will be discussed and users can present and share their data, troubles, and achievements. The next meeting will take place on the 7^th of June 2017 where we will discuss single cell sorting tips, tricks and considerations. If you have any topics that you would like discussed please let us know!

@WRHflow is now on Twitter

Follow us on our new twitter feed for the latest updates to the WRHflow facility!

ACS2017

CYTO Asia will be held in Singapore from the 22^th to the 27^th of October. There are 10 x travel awards available to Australian members to get to the conference. I strongly advise for all students doing cytometry related work to apply for these awards. To apply you must submit an abstract, your CV and complete the applicable travel award application. We will forward more information as it is available.

Oral and Poster submission deadline: 24^th June.

For more info visit: https://www.cytometry.org.au/content/cyto-asia-singapore-2017

ACS Clinical Roadshow 2017

Following the success of last year, ACS will be holding the Roadshow on the 28^th of June at the Royal Prince Alfred Hospital. The Focus this year will be on Clinical Flow Cytometry and this full day seminar will be valuable to all interested in clinical and diagnostic flow cytometry.

More information can be found following this link:

http://www.cytometry.org.au/content/acs-roadshow-2017-sydney

Reagents available from WRHflow

To assist researchers in panel design and experiment outcomes we have reagents available for immediate pickup via orders placed through PPMS. Items include: DAPI, BD Brilliant Stain Buffer, Human RBC lysing solution, FACS tubes with cell strainer caps, GentleMACS tubes, with other reagents to be added as they arrive.

Upcoming tutorial session run by WRHflow – 29^th June 2017

WRHflow will be holding a tutorial session aimed at both beginner and advanced flow cytometry users on the 29^th of June. The session will be split into two sessions, one aimed towards new users, focusing on key concepts and setting up the instrument, followed by an advanced session focusing on flow cytometry tips and tricks to ensure the generation of quality data and data analysis. Keep an eye out for the notices. 

Fortessa baseline change

From 2015 to the current, we have maintained the same target MFIs on the BD Fortessa checked via an almost daily quality control. This has allowed users to utilise application settings for longitudinal studies. Unfortunately, the detectors off the UV laser have slowly been reporting reduced sensitivity on and off for the last month and after extensive engineer efforts we will need hardware replacements. We are currently planning on replacing the fibre optic and/or the PMT detectors to increase the sensitivity to a level where it previously was. Hopefully resulting in increased sensitivity and the instrument not intermittently failing QC.

To those users using application settings on the BD Fortessa, and wish to continue with existing values, know that you will need to save new application settings moving forward as we will reset the baseline on the 23th of May once the detectors are replaced. We expect minimal changes in other FL detectors but PMT voltages may need to be adjusted off the UV laser detectors to maintain similar MFIs in your populations.

The best means to do this would be to match positive bead signal intensities of all FL detectors, with previously recorded data files, especially CS&T beads or another QC bead, on an experiment post the 23th of May, and then saving new application settings. This does mean looking at a statistic and matching the recorded signal with current signals.

Another method that would work for users who have not recorded beads in prior experiments, would be to switch to the existing baseline (the one that existing application settings were saved on), run a CS&T, make a new experiment, apply existing application settings, copy the voltages, switch back to the new baseline, make a new experiment, copy the voltages over to the new experiment, and save new application settings. Of course, this will cater for all FL detectors (and will be the best possible means otherwise for the UV). If any help is required please contact WRHflow. 

Refresher - How to run CS&T (Q.C) on the WRHflow analysers

Before running an experiment, you should always check if CS&T has been run, by looking at the survey that pops up once you log in to the PC. If it hasn’t been performed, for example on a weekend, we recommend you will need to run it yourself as trained. Briefly this can be done by following these steps:

Making CS&T beads (if not already made and labelled in the flow fridge):

• Add 300ul saline into a FACS tube.
• Vortex the CS&T bead stock vial for 30s, add 1 drop into the FACS tube.
• Vortex briefly before use.

CS&T on CantoII:

• Turn on the instrument and computer, allow 30min warm up.
• Log in & launch FACS Diva.
• Go to Cytometer -> Fluidics Startup. This will take ~7min.
• Go to Cytometer -> CS&T.
• Load the CST tube and click run. This will take ~10min.
• Following the run, check that it’s passed (if it hasn’t passed this indicates a potential problem with the cytometer), select use CS&T settings when you leave the CS&T panel.
• The instrument is now Q.C’ed ready to use.

CS&T on LSRII & Fortessa:

• Turn on the instrument and computer, allow 30min warm up.
• Log in & launch FACS Diva.
• Prime the instrument a couple of times.
• Run H20 for at least a minute through the SIP to remove bubbles from the prime.
• Go to Cytometer -> CS&T.
• Have flow rate on low, and the fine adjust knob 5 full rotations from either end.
• Load the CST tube and click run. This will take ~10min.
• Following the run, check that it’s passed (if it hasn’t passed this indicates a potential problem with the cytometer), select use CS&T settings when you leave the CS&T panel.
• The instrument is now Q.C’ed ready to use.

Account Syncing Across Analysers / New Analysis Template Added

On the 29^th of May we will sync BD Diva accounts across all flow cytometer analysers (2xCantoII’s, LSRII, Fortessa). This will mean that all instruments will have the same available accounts listed below. The password for each account will remain as the username (in lowercase letters). A basic analysis template with population hierarchy will also be available on all instruments. Select it while creating a new experiment by choosing “Experiment” > “New Experiment” and under the general tab selecting “Generic WRHflow Template” under the general analysis template tab.

A little bit about using MFI, GMI, % in flow data analysis

Mean-FI (mean fluorescence intensity)

Med-FI (median fluorescence intensity)

GMI (geometric mean intensity)

% positive

Firstly, since MFI is used as the acronym for both the mean, the median and even the geometric mean of fluorescence intensity, ensure you know which statistic you are actually dealing with.

Most flow data is acquired in log, so you should use the GMI over the MeanFI, as it will give you a better indication of the centre of the data, whereas the MeanFI can be easily influenced by outliers. A few outliers can greatly increase your MeanFI, whereas a GMI or a MedFL is affected less so, see below.

Since flow data is seldom normally distributed you should use the median as it is more robust and less prone to skewed distribution or outliers. Median is a good representation of a "typical" event.

For skewed data, it is important to understand why the median is a better choice as it provides some description of the underlying data, whereas in the below example, the mean of the skewed data does not.

For bimodal distributions, neither mean nor median will be informative. In this situation a combination of % positive/negative and MedFI/GMI is a better choice. Understanding what is happening in the below plots is quite useful. If we look below all that is happening is the frequency of the positive population is increasing from left to right, with the negative population staying the same. Notice how misleading, and no-informative the MedFI, MeanFI and GMI can when describing bimodal populations? I have included separate statistics on the gated negative and positive populations to show the statistics on the gated populations.

And lastly, correct interpretation of the below data is important in understanding the levels of expression. All below samples have approximately equal positive population frequencies of ~60%, however the level of expression of the marker in U740_35A can be reported as different between samples (increasing left to right).

BD Antibody Recall

BD recently initiated a voluntary recall for a subset of Research Use Only (RUO) single vial reagents with fluorophores. This recall was initiated after observing sporadic performance and vial to vial variability during routine internal testing as well as through complaint investigations. BD has completed a thorough investigation and evaluation of product inventory of Lots shipped between May 16, 2016 and February 10, 2017. Based on those outcomes, our initial notification is being expanded to include additional lots of RUO single vial reagents with fluorophores.

A description of the issue and impacted product formats are listed on our website www.bdbiosciences.com/productnotice. Locally we have reviewed these outcomes and determined your institution has been shipped affected product. BD recommends that you either:

1. a) Discard any remaining inventory and request a credit or b) Perform quality control using biological cells per your standard protocol on each vial of reagent to confirm that the reagent is still performing as intended. c) Please also review any data that may have been generated from any affected product. The potential degradation of product may result in a false identification of cell populations and/or aberrant staining patterns.

If you have any queries please contact either Nikki Alling (0408549003) or Hayley Suen (0436 043 636).

Monthly User Group Meetings *update - schedule will be available via intranet*

Starting Wednesday the 3^rd of May – 2017, followed by every first Wednesday of the Month - 4.00pm-5.00pm

User group meetings will be held in the Level 2 Conference Room C2.31 (followed by conference room C2.20) where flow cytometry topics will be discussed and users can present and share their data, troubles, and achievements. The first user group meeting will discuss the spill over matrix, instrument specific stain indexes and reviews of current working panels. The meeting will take place on the 3^rd of May 2017.

Cell Sorter Data Access

User data generated on the cell sorters can now be accessed (within WIMR) via the Scientific Platforms network drive. The address to map the drive is \\wmi-fs03\scientific platforms\flow cytometry\sorters\

Please note data generated on the Influx is available immediately, however data generated on the ARIAIII will be available the following day after a sync. Non-WIMR users will be able to access their data via a Cloudstor account that is synced with the server.  

New Cytometry Specialist Technician

Please make Edwin feel welcome to WIMR. Edwin’s background is in B cell immunology where he was researching somatic hypermutation mechanisms using an animal model. He has experience in cell sorting for various downstream applications including adoptive cell transfer, single cell PCR and microarray. He also has experience with a range of flow cytometry applications such as cell proliferation tracking & cell cycle analysis.

How long can I book the analysers for?

We are continuing with the current booking policy where the flow cytometry analysers can be booked for a maximum of 2 hours during business hours (8.30am-5pm). However, from 3 o’clock users can book slots as long as necessary. This policy enables multiple users to acquire samples especially during the peak hours that cover midday to 5pm. Please let us know what you think, or whether you have any suggestions. We will constantly monitor and review the policy and reconsider the policy in the future if necessary.

BD Horizon Multicolour Workshop

We will be hosting a BD Horizon Multicolour Seminar and Workshop at WIMR on the 4th and 5th of April, 2017. The seminar will be held in the Conference room 2, C2.22 @ Westmead Institute for Medical Research on Tuesday, 4^th of April, 9:30am - 1:00pm. The practical workshop, which will be held on the 5th of April, in the flow labs, has limited spots and is now full. Registration for the seminar are through the below link. These workshops have been very well received and useful in previous years. If you can attend, I would highly recommend to do so.

https://www.eventbrite.com.au/e/bd-horizon-multicolour-workshop-tickets-32104945757

Updated autoMACS shutdown procedure

The autoMACS, an automated, magnetic cell enrichment separator, installed in late 2015, has been steadily gaining use over the last 12 months. To prevent any system contamination, a new shutdown procedure has been documented. It can be found on the front of the instrument biological safety cabinet and takes ~10 minutes to run. In addition to a regular maintenance and cleaning schedule, this should ensure contamination free future operation.

Reagents available from WRHFlow

To assist researchers in panel design and experiment outcomes we have reagents available for immediate pickup via orders placed through PPMS. Items include: DAPI, BD Brilliant Stain Buffer, Human RBC lysing solution, FACS tubes with cell strainer caps, with other reagents to be added as they arrive.

FluoroFinder panel design

To assist with an increased number of queries regarding panel design on the flow cytometers we have registered our instruments with a leading cloud based panel design solution that was well received at CYTO2016. We have recently updated our cell sorter configurations on FluoroFinder in addition to our cell analysers. FluoroFinder can help you design panels specific for our cytometers, help compare spectra from multiple vendors, and save time in panel design or at least, be used as a guide to a working panel. 

To get started head to https://app.fluorofinder.com/ We are now a registered shared resource lab. You will eventually need to make an account which allows you to save all your panels (it’s free for users)!

Upcoming seminars run by WRHFLOW

In addition to the BD Horizon Multicolour Workshop, we will be holding a seminar aimed at beginner flow cytometry users in the last week of April with a date TBC. The seminar will be aimed towards new users, focusing on basic tips and tricks to ensure the generation of quality data. The seminar will also cover the basics of FlowJo analysis. Keep an eye out for the notices. 

INFLUX & ARIAIII upgrade

The cell sorters were upgraded with additional parameters over the new year shutdown period. The Influx now has 24 parameter capability (up from 22) and the ARIAIII now has 16 parameter capability (up from 12). The configurations were selected to utilise newly released dyes, maximising the UV and Violet laser lines. The current configurations can be found at the end of this newsletter.

Luminex 200 added to PPMS

The Luminex 200 has been added as an instrument that can be booked using PPMS. This will help to ensure no booking clashes occur between users. This will also provide usage statistics in order to efficiently manage the ordering of calibration/verification kits that are necessary for proper operation.

*Could existing users who have been trained please email wrh.flow@sydney.edu.au with the following information – Name, Date trained, Trainer, so that we can add you onto the registered user list.

How to use Application settings on BD FACS DIVA – FOR NEW EXPERIMENTS

Application settings are useful in ensuring longitudinal studies (studies measured over multiple days/weeks/months) maintain consistent MFIs over the course of the experiment. We have successfully implemented and assisted many users in creating and applying application settings to their experiments.

The following workflow can be used to successfully save and apply application settings. Please note this is only one workflow to save and apply application settings. There are several variations that could also work (and some that might not). We are covering 1 specific workflow today. The 2 key points to remember are, 1, that you check that voltages have updated to ensure successful application of application settings, and 2, to record data for beads (CST, with bead lot recorded are good for this, or other long term QC beads may be used) before and following your experiment, every day you collect data. Running these beads allow continuation of targeting similar MFIs, even if the instrument undergoes major change, or if the baseline settings expire for application settings.

The below listed protocol is for creating a new experiment with N compensated parameters. It assumes you have 1 panel with N flurochromes, and will setup and apply a compensation matrix to your data when starting. The assumption is that you have compensation controls for all parameters. This protocol creates application settings that are used on subsequent days to ensure consistent MFI’s. We will be documenting a different protocol for setting up application settings with an exported experiment template as there are slight variations.

PROTOCOL – Setting up a new experiment utilising application settings.

Instrument Setup

• Ensure CST has been run on the instrument for the day, with the current bead lot number in use (can be checked via the instrument log that displays automatically upon logging in to the instrument PC).
• Create a new blank experiment under your folder.
• Label the experiment with a descriptive name following the syntax ‘YYYYMMDD_Descriptive_Name’.
• Click ‘Cytometer Settings’
• Check the FSC-W, FSC-H, SSC-W, SSC-H parameters for recording in the Inspector tab as below to allow identification of doublets.
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• Click > ‘Menu’ > ‘Experiment’ > ‘Compensation’ > ‘Create compensation controls’.
• Delete unused parameters. Leave the compensation tube label as ‘Generic’. Ensure you have ‘Include separate unstained control tube/well’ checked at this point. Click OK when complete.

• Activate the ‘Unstained Control’ (as it has a useful default tube specific workspace that shows all parameters).

Minimal Detector Voltage Setting

• Acquire unlabelled cells. Adjust FSC and SSC voltages as needed to bring populations onto scale when looking at a FSC/SSC bivariate plot (take note of the voltages needed for cells).
• Move the already drawn gate, P1, around the cells of interest. Right click a bi-variate plot and click ‘show population statistics’. Edit the statistics display to show the rSD for each fluorescent parameter used (see figure below). Record enough cells to determine the rSD of P1 for all parameters that will be used. You need to record a small file in order to accurately determine the rSD of the unlabelled cells.
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• Adjust PMT voltage to bring the rSD of the unlabelled cells to 2.5x the value of the rSD of electronic noise (that is viewable from the instrument configuration sheet) that accompanies each cytometer. You need to record a small file in order to accurately determine the rSD of the unlabelled cells each time.
• Take note of these PMT voltages. These are the minimum PMT voltage that should be used for each detector for optimal detection of dim epitopes.
• At this point you now have the minimum voltage for each detector needed to separate dim from negative signal.

Compensation Controls

• Now, while the ‘Unstained Control’ is still activated, we will acquire data from the compensation controls. Adjust FSC and SSC voltages if needed to bring populations onto scale (take note of the FSC & SSC voltages needed for beads, if used).
• Sequentially acquire data from each compensation tube. In each, adjusting the PMT voltage for the fluorochrome if necessary, ensure that the positive fluorescent signal is on scale and within the linear range of the detector, ensuring the signals are using the full dynamic range of the detector.
  • Setting of the voltage is to ensure that the resolution between dim and negative events is best resolvable and that highly positive cell populations still are within the linear range for each detector..
  • Remember there is a range of optimal PMT voltages that will provide similar resolution between dim and negative cells.
  • PMT voltages that are too high will not affect the resolution of dim and negative cells, but will unnecessarily cause spreading of the negative population.
  • Any bright populations with very large CVs (very broad distributions) must have their PMT voltages lowered so that the brightest events or MFIs remain within the linear range of the detector.
  • Remember your compensation controls should be representative of your samples, ensuring they are as bright or brighter than your sample (but not log folds brighter preferentially).
  • Keeping positive signals on scale (and within the linear range of the detector) overrides the rule for increasing the PMT voltage to target 2.5x rSD of electronic noise.
  • Remember to leave enough room above the positive signal, and in the linear range of the detector, to allow for increases in fluorescence of certain epitopes
• So, at this point, while the ‘Unstained Control’ in the compensation controls folder is still selected, you should have cycled through your unlabelled cells, single stained compensation controls, and a little of your sample (to check everything is on scale and as expected).
• After the above steps, your voltages should now be set. We can now record compensation controls. Do not change any fluorescent PMT voltages, it is okay to change FSC and SSC detector voltages to those noted as you acquire different cell and beads for your compensation controls.
• If you are not using a universal negative, at this point, modify the compensation controls and uncheck the universal negative compensation control. If you do the afore mentioned, ensure to create a P3 gate in the tube specific worksheets for each parameter to be compensated as the software looks for a positive signal in the P2 gate and a negative signal in the P3 gate (TIP: remember you can append data if you have only positives in a tube and only negatives in another tube for non-universal negative compensation controls). If you are using a universal negative compensation control, proceed and record compensation control data without modifying the compensation controls.
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• Calculate the compensation matrix, and click ‘apply and link’ (this saves a copy of the matrix into FACSDiva).
• Give the compensation settings a name following the syntax ‘YYYYMMDD_Descriptive_Name_Comp’

Experiment Setup

• Make a new specimen and tubes as necessary.
• Menu > ‘Experiment’ > ‘Experiment Layout’
• Label all parameters, setup acquisition parameters, storage gates, event numbers, and input keywords.
• Rename specimens / tubes as needed.
• Setup workspace layout as necessary. A good workspace observes all parameters in strategic bivariate plots, and monitors the acquisition over time by drawing a parameter using last laser on the Y axis with Time on the X axis. A good experiment will also have a tube where the user can record beads (QC beads, 8 peak, CAB beads, etc.) both as the first tube and the last tube in the experiment, ensuring that there was no aberrant instrument occurrences during the acquisition and also to allow adjusting of PMT voltages in the event of instrument component replacement or instrument baseline update.
• Load your first sample to be acquired and recorded. Activate the tube in the browser. Acquire data, and quickly adjust the FSC / SSC detector voltages to those noted for cells. Record your first samples.
• Right click the top-level cytometer settings and click ‘Application Settings’ > ‘Save’.
• Give the application settings a name following the syntax ‘YYYYMMDD_Descriptive_Name_AppSettings’.
• Acquire and record data for each sample.
• Export data.
• Note down all experiment details.

 Restoring application settings (from the original experiment, not from experiment template)

• Open the original experiment (for this protocol, do not open an experiment that has already had application settings restored, only open the original experiment).
• Right click experiment > ‘Duplicate experiment without data’.
• In the newly created duplicated experiment, right click the duplicated experiments top-level cytometer settings ‘Cytometer Settings’ > ‘Application Settings’ > ‘Apply’ > Apply saved application settings
• As the copied experiment will have a compensation matrix that is non-zero from when the experiment was setup, click ‘keep the compensation values’ (you can re-record compensation control tubes and recalculate the compensation matrix if desired).
• If a dialog appears asking to ‘Confirm Cytometer Changes’ – in regards to FSC Area Scaling values have changed – overwrite them by clicking YES.
• If you are not recording new compensation controls, proceed to record samples (adjusting FSC and SSC detector voltages to those noted if necessary).
• If you are recording new compensation controls (recommended), right click the compensation tubes ‘Cytometer Settings’ > ‘Application Settings’ > ‘Apply’ > Apply saved application settings. Record compensations (only adjusting FSC and SSC detector voltages (as noted) to bring populations onto scale, then calculate compensation. Clicking apply and save.
• Check the voltages have been slightly updated.
• Proceed to record samples (adjusting FSC and SSC detector voltages to those noted if necessary).