8 Peak Beads – Now available to users
8 peak beads are now available for users to acquire as a means of QC on the WRHFlow cytometers. Users can find the 8 peak beads alongside the QC(CST) beads in the flow cytometry fridge in the shared lab. Acquiring 8 peak beads before and after your acquisition provides information on whether the instrument has changed from the start to the end of your run. They can also be used to set/monitor PMT signals between acquisitions performed on different days in synergy with application setting for those users who are using application users. Please ensure you record the bead lot number!
Instrument Baselines – November 2018
From 2017 to the current, we have maintained the same target MFIs on the 2 CantoIIs and the Fortessa via an almost daily quality control (note: LSRII baseline had been updated earlier in 2018). 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 current QC beads in use will finish in the next couple of months we are required to establish new target MFIs (also known as baselines) for the new bead lot that has arrived. This is also necessary when major changes have occurred on the instrument. Major notable changes that have/will occur include laser, computer, and electronic component replacements on different instruments.
To those users not using application settings there will be no further disruption.
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 2018 as per the below schedule. 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. 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 #70466 (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 #80998 (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.
It is important to ensure the above process is completed once started – to ensure the most recent CS&T report is from the #80998 bead lot, as the instrument uses the last CS&T data acquired.
Unfortunately the above method will not be applicable to the LSRII as we are planning a computer/software change. LSRII users are recommended to run and record 8 peak bead data (and record the bead lot) referencing peak locations in order to carry over any detector voltages if necessary.
New Baseline Implementation Schedule
Instrument | Date Scheduled | Bead Lot |
CantoII_ICPMR | 5/11/2018 | #70466 > #80998 |
CantoII_WIMR | 12/11/2018 | #70466 > #80998 |
LSRII | 31/10/2018 | #70466 > #80998 |
Fortessa | 19/11/2018 | #70466 > #80998 |
Symphony | 17/9/2018 | #70466 > #80998 |
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.
FlowJo Basic and Advanced Tutorial – Save the date!
November 1st 2-5pm – WIMR Seminar Room C2.20.
We will be hosting Jack Panopoulos – FlowJo Application Scientist – as he delivers updates on basic and advanced flow cytometry data analysis using FlowJo & SeqGeq. There have been a number of recently released plugins that there will also be focus on that should interest many.
NanoFCM demonstration – Get your 7-500nm samples ready!
September 19th & - October 15th – WRHFlow Labs - J2.05
Small particle detection/characterisation using flow cytometry has been a hot topic in past years. A number of researchers here are currently focusing on nanoparticles/microparticles/viruses. The WRHFlow team have been interested to see how a ‘novel’ flow cytometer / technology performs in this space of <500nm and therefore have organised a demonstration/trial of the NanoFCM.
We will have company engineers on site who can assist running samples for the purpose of the instrument evaluation on the 19th September 2018. We will then have the instrument for ~1 month for evaluation – with assisted sessions available to researchers to evaluate the instrument. Samples will need to be run by WIMR staff.
We will hold a small presentation from 11am-12pm on the 19th on the L7 meeting room to gain an understanding of the technology that you are welcome to attend.
For access, please see reception if needed.
Sample acquisition is slated for the afternoon - ~12-3pm and we welcome your samples for testing.
Please note as this is an instrument evaluation user data may be used to furnish a study report of instrument applicability. Also any data will need to be provided to NanoFCM for review prior to publication. If you are interested in the instrument I would suggest evaluating it with a sample of interest, especially while the engineers are here on site. Further instrument information can be obtained here - https://www.nanofcm.com/en/Product/NanoFCM/117.html
BD Symphony – 8 laser, high sensitivity flow cytometer now installed, training for high parameter panel users.
August 22nd - 11am & 2pm - J2.07
September 4th – 3pm – J2.07
September 21st - 3pm - J2.07
Please RSVP with your intention to attend to wrhflow@sydney.edu.au
View configuration_V1 for panel design here
View configuration_V2 for panel design here
CantoII HTS installed – Time to think about preparing samples in 96 well plates?
All our flow cytometers have now been upgraded with high throughput capabilities. That’s a total of 5 high throughput sampling robots in the flow cytometry facility.
Benefits of sampling from a 96 well V bottom plate include minimised loss of cells between washes, 8-12 samples at a time using a multichannel pipette, better washes as virtually all buffers are aspirated in a 96 well V bottom, and robot acquisition!
If you require training for using the HTS please submit a training request through PPMS.
If you would like a 96 well V bottom plate, these can be ordered through PPMS for $6 each.
BD Symphony install - First Symphony A5.2 (not A5.1) install worldwide!
You may notice a number of engineers in the flow labs this week with the BD Symphony installation/testing underway. Geoff Osborne (BD SORP director) and the team will be busy installing the world’s first updated A5.2 which has a revised detector configuration and new electronics.
We have scheduled in two initial training sessions on the 22nd of August 2018 @ 11am & 2pm (with others to be announced). If you would like to attend this initial training please RSVP to wrhflow@sydney.edu.au for these sessions (we will limit them to 5 each session).
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Failed laser teardown!
Curious what happens inside a diode laser like the below used on the Fortessa? We were un/lucky enough to have had the red laser fail on the Fortessa in August 2018 and so we did a laser teardown.
Lasers come in many shapes/sizes/forms. A very popular choice these days are diode lasers. They are cheap, reliable and come in a range of laser power outputs. The red laser on the Fortessa was one of these.
Solid state laser - Coherent CUBE 639nm 40mW laser
These red lasers have a 'cleanup' bandpass filter to ensure only ~639nm light exits (the little circle lens), and a physical shutter operated by the lever (right)
Simple to open (but tricky, don't forget the hidden screws under the CUBE sticker). The unit pulls apart easily. The main control circuitry, diode, and heatsink are positioned on a nicely CNC'ed solid block of steel (middle).
A close up of the motherboard and diode.
The various boards pull apart easily. We can see the communications board - with the USB connection. The board with the golden connector is for modulating the output. The other circuitry needed for a stable laser include a temperature / emitted light / current draw sensor and control logic. Next to the diode laser is the thermal controller for the heatsink (it can heat and cool the diode). The beam shaping prisms can be seen mounted on the heat sink. This model produces an elliptical beam.
The laser had way more than the average life expectancy for these lasers. This laser had >10000hrs and the diode had failed.
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2018 Tips and tricks competition winners announced!
Congratulations to Kirstie Bertram and Nicole Fewings for winning the WRHFlow Tips & Tricks competition.
Kirstie’s entry highlight a number of important points to consider including antibody clone / manufacturer differences when selecting antibodies for a panel, while Nicole’s entry demonstrates the benefits of titrating not only antibodies but viability dyes. See the/wiki/spaces/DMT/pages/400916724for winning entries.
Instrument bookings...
In the last 6 months the 4 flow cytometry analysers located at WRHFlow have seen over 100 different users resulting in over 2000 hours of actual collective usage, with about 1/4 of usage being after business hours. These numbers are wonderful and depict an increasing usage of the instruments.
There are 2 statistics however that are of considerable interest to me.
41% of the time booked on instruments is not used.
50% of the time used is not booked.
...
Image Cytometry
‘delightfully detailed dots’
I recently had the opportunity to acquire samples using an image cytometer for a research project using an ‘Imagestream’, accessed via Sydney Cytometry at the CPC. An image cytometer will generate images for the events that are collected. The instrument I used outputs not only multiple fluorescent high quality images that can be matched to specific dots in traditional flow cytometry data, but provides automated image analysis modalities for data mining. Despite the release of initial variants of the technology way back in 2004, it is an often overlooked and underutilised tool in the cytometry toolkit. The spatial localisation of fluorescence signals is incredibly powerful in the addition of features (similar to parameters in traditional flow cytometry) for sub setting populations. If you are interested in image cytometry please contact westmead.cytometry@sydney.edu.au to understand how this technology may add to your research and how you can access the technology.
Levitas LeviCell Trial – ITS HERE NOW – February 22nd to March 5th 2021
‘live cell separation using microfluidics, magnets and MRI technology’
Westmead Cytometry will host the LeviCell for approximately 2 weeks for interested researchers to trial the technology. The small instrument (0.7mx0.3m) essentially allows for the rapid bulk isolation of live cells from debris and dead cells. There has been considerable interest for the potential of this instrument to slot into workflows where cell dissociation from tissue samples are a necessity. An evaluation of the system will follow in the next newsletter but if you would like to evaluate the system please email your interest as there is availability but reagent limitations.
Unused Analyser Session Charges
Starting on the 1st of March 2021 (to be delayed and now with 3 strike rule in place), booked but unused sessions on the analysers will be charged at 25% of the hourly rate as per the published 2021 Westmead Research Hub user fee schedule to discourage users reserving instruments during peak periods with usage not occurring. The charge is primarily for users who unfortunately book but do not show up resulting in a disadvantage to all users.
Westmead Cytometry User Group Meetings
‘2nd Friday of the month @ 11am
A forum to communicate with fellow cytometrists in an informal, friendly and ultimately thought provoking environment. The user group meetings will consist of a small delve into a research project/topic followed by an open session with the aim to improve and discuss researchers concerns right from sample preparation to data analysis. The schedule for 2021 is included below and will be held in the level 2 WIMR seminar rooms.
Fluorochrome Degradation & Compensation Options
‘validation is key’
I thought it useful to outline (at least on the instruments at Westmead Cytometry) different approaches users are implementing when deciding on the practicality of generating for high parameter compensation controls.
Our recommendation is to generate compensation controls with fluorochromes that have undergone the same process as your sample (and in parallel), followed by validation that the spectral profiles of the compensation controls does indeed match the spectral profiles of the flurochromes used on samples acquired (by ensuring populations are not over/under compensated, along with identification of intended populations).
However with the increase in high parameter cytometry popularity and practical/financial constraints there have been variations to the above recommendations observed. These variations include
-fixing compensation controls with acquisition on subsequent days
-unfixed compensation controls with acquisition on subsequent days
-utilising a compensation matrix across different days with no application settings applied (and 8 peak instrument checking)
-utilising a compensation matrix across different days with application settings applied
-acquiring samples on days after staining with compensation controls made on the day of sample acquisition
etc.
It is important that in the above listed variations that the compensation matrix applied to the samples is validated not to change populations identified. The good news is that the validation is quite straightforward and generally requires the acquisition of the same compensation controls at different timepoints. In addition, a change in the matrix does not necessarily mean the entire set needs to be remade. There have been cases where antibody conjugated fluorochromes were stable but viability markers were not, and other examples where the switch of certain fluorochromes from beads to cells resolved the changes observed on subsequent days.
The take home message is variations to the recommended workflow are occurring and as long as validated to not alter the matrix applied to the sample causing population changes then they are valid approaches.
Fancy Using A Supercomputer? I did (and loved it).
Argus is the University of Sydney’s “Virtual Remote Desktop” project designed to bring remote, on-demand, interactive, graphically intensive compute environments to researchers.
Westmead Cytometry & Westmead Imaging now provide access to 3 of these systems for you to access for those compute intensive applications. These remote desktops are fast. Each system has 16x XEON core CPUs and an impressive 128GB RAM alongside 8GB GDDR5 NVIDIA GPUs with network throughputs of 200+ GBPS. NBN is 1GBPS for comparison.
The Argus team (special thanks to Ashok Pachipala @ USYD) have installed all of the right applications including R, RStudio, Huygens, ImageJ, CellProfiler, FlowJo(T&Cs apply), Office, AutoCAD and others making it an excellent desktop to log into, setup something that will take a while (tSNE, Image Deconvolution, etc), and let it do its thing.
Access is easy, all that is required is a Unikey and a return email acknowledging some basic guidelines. Booking is via PPMS and shhhh… they aren’t charged. These desktops have proven so capable that they even have altered mindsets of purchasing new analysis PCs.
Have Something You Need *3D* Printed?
A reminder that we have a 3D printer that can be accessed by all researchers. It has proven very capable with a number of useful prints having been produced to date. Slide holders, electron microscope parts, centrifuge parts, kidney transplant mounts, and a build your own cytometer. If you have a requirement for a custom designed novel scientific part that will assist your research, let us know!
More information can be found here
An Automatic Plate Washer!
It has been brought to my attention that many researchers are in the dark that we have an automatic 96 well plate washer available in the flow labs. If you could use this for plate based assays, such as ELISA’s, or protein quantification assays, then it is very easy to use. If you would like to be trained to use it training requests are through PPMS.
For a complete list of instruments available at Westmead Cytometry click here
Cell Counter - Now Available – Give It A Try.
Still counting PBMCs or splenocytes manually?
The cell counter can also. 50uL and ~<1min/sample.
The newly installed CytoFLEX (2 laser, 4 parameter) flow cytometer is available to use @ Westmead Cytometry. The instrument can be used as an accurate cell counter providing researchers with an accurate, quick, accessible cell counter to utilise in your research. The instrument doesn’t require booking and training is via key superusers located across the WRH. In addition to cell counting, parameters such as GFP expression, viability and scatter profiles can be collected. The instruments accepts Eppendorf tubes and 5mL FACS tubes, requiring 50uL and ~40seconds per sample. Facility provided DAPI and Syto Dyes are available also.
Key SuperUsers who can be contacted for training/further information include:
Suat Dervish
Edwin Lau
Ya Wang
Joey Lai
Cindy Zhu
Stephen Schibeci
Daniel Hu
Nicole Fewings
Cyto2019 – Vancouver - Abstract Submission Is Now Open
Cyto is unquestionable the most prominent cytometry research conference internationally. Cyto2019 will be held in Vancouver, June 22 – June 26 – 2019.
Submit an abstract before February 11th for your opportunity to present your data to an audience of cytometry/immunology/many discipline experts (an eclectic bunch). Just as important is what can be gained from the conference, i.e. the latest in data analysis workflows /algorithms, latest reagent/assay information, and sneak peaks of novel disruptive technologies entering the cytometry space. Travel awards are available for eligible applicants.
Scientific Platforms Data Clean Up Reminder
Some of you are probably using the Scientific platforms drive to store you exported flow data temporarily. As the drive is getting full, we will have to clean up the drive according to our data policy: All data >14 days old will be deleted.
On 15/2, IT will be deleting data files on Scientific Platforms that are more than 14 days old automatically. Please save all wanted files onto your local storage.
If you have any concerns please contact us.