CytoFLEX Cell Counter / Flow Cytometer to be installed installed Tuesday 30th October 2018!
We will be installing the recently acquired CytoFLEX next week and will soon update on access guide to using the new instrument. The CytoFLEX provides volumetric acquisition providing cell counting to users.
Training for key SuperUsers will occur on Friday 2nd November with an access workflow finalised soon after for users to start using it.
Left: Cytoflex Right: Ian & Mathi post Cytoflex install
Christmas Closedown
The Westmead Institute will not be closing over this year’s break; please note there will be less support services and reduced building access hours from 8am-8pm each day.
Please note that Westmead Cytometry will not be serviced from Monday 24th December 2018 (Christmas Eve) to Tuesday 1st January 2018. 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 21st of December as they will be shut down for the Christmas Closedown. The sorters will also not be open for bookings on Wednesday the 3rd of January as they will be brought back online on this day. The first available day for a sort for researchers will be the 3rd 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.
'WRHFlow' to transition to 'Westmead Cytometry' in 2019
We will migrate to using westmead.cytometry@sydney.edu.au over the next few months but will continue to monitor both the new email address as well as the existing wrh.flow@sydney.edu.au email address.
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.
*Note for CantoII & LSRII you will need to change to an existing configuration. Please contact staff if help is needed.
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.
Please users, remember to cancel sessions that can not be used as soon as possible, as this will undoubtedly assist other users who may be staying later to run samples. This will also allow WRHFlow to remain not charging for unused booked time. The latter which occurs at other facilities.
Fortessa HTS upgrade!
The Fortessa now has the hardware required for acquisition from a 96/384 well plate. We have organised an en-masse training session on the 12th of July @ 9am for any one who would like to use it.
Data / Information / Spotify MAC now in LSRII/CantoII lab
Browse the internet for cytometry related information, click through the WRHFlow homepage or listen to tunes in Spotify while acquiring data in J2.07.
Informal flow cytometry data analysis - 26/7/18, 4pm, WIMR conference room C2.20
Like the plugins available in FlowJo, interested in flowCore in R, fancy t-SNE plots...
Bring a notebook, some data, and let's collectively contribute to the data analysis confluence page while hopefully learning a trick or two from peers. This will be an informal get together where users can ask, contribute or add wisdom about anything flow cytometry data analysis related.
Session to be confirmed!
Tube loader & high throughput sampler installations on all cytometers
We are planning the installation of an automated tube loader on the CantoII as well as high throughput samplers on the Fortessa and CantoII over the next 3 weeks. We will be booking ~4 hours on each instrument to complete the installations and are very excited to see these installed. Keep an eye on the latest news page to see the install.
Notice - SIP sheath for the LSRII and Fortessa
The LSRII and Fortessa must have SIP sheath covers installed to prevent dripping of samples.
- The SIP sheath cover is installed by default on both instruments.
- The SIP sheath cover can be removed (but must be reinstalled immediately for a different sample or following your session) for the following samples only if -
- Bead samples are immediately to be acquired, OR,
- The samples have been inactivated by fixation. An example protocol for sample fixation could consist of 20 minutes exposure @ 4°C to a flow cytometry specific commercial fixative containing paraformaldehyde (example buffer set & protocol). Other protocols that result in fixation of samples are also valid, OR
- The samples are being acquired from a HTS device.
- A properly installed SIP sheath cover will look slightly thicker than without the SIP sheath (compare with below photo) and should not drip liquid when the instrument is on run. To remove the SIP sheath on the LSRII or the Fortessa follow these instructions. To replace it, the order is reversed.
- To reinstall the SIP sheath cover follow the reverse instructions. The above applies to the LSRII & Symphony.
A tweaked data export workflow for users
WRHFlow have instigated some automatic data syncing scripts for users who would like to try them out. Unless your data should not be synced to the WIMR server, when exporting your data, export it as FCS3.0 format, with linear format selected to 'D:\BDExport\FCS' (as this folder will from now be automatically synced to the WIMR server). If you would like to copy your data elsewhere you can also copy it from this location. Your data should be able to be accessed from the Scientific Platforms networked drive within WIMR, and via a username/password for non-WIMR users via Dropbox/Onedrive if desired (email wrhflow@sydney.edu.au to setup an account).
Drop off your sample for multiplex analyte analysis?
We are considering implementing a core facility run multiplex assay service to be run on the Luminex 200 to enable users with fewer than 76 samples to merge with other researchers to complete a 96 well kit that is used for multianalyte analysis of chemokines, cytokines, growth factors, etc.
Users would register a spot on a 96 well plate for a particular day. There would be 76 available sample locations as 20 are reserved for standards and QC. On the day, users would drop off their samples, on ice & labelled to the flow cytometry lab by a certain time. WRHFlow would then centrifuge samples, load them, and follow the protocol using automated means followed by plate acquisition on the instrument. The data would then be disseminated to researchers.
We would like to collect feedback that if this was a service provided would it be utilised by you or your group, please let us know via this feedback form over the next 2 weeks.
16th-18th July 2018 - Oz Single Cells 2018
≤September 2018 - New 50 parameter instrument to be installed at WRHFlow
The UV laser has changed on the Fortessa
See here for continuing use of application settings
Student training gets a boost - Cyto U access
The International Society for Advancement of Cytometry (ISAC) is dedicated to advancing cytometry through a number of different educational programs. CYTO U is an online portal available to ISAC members that provides peer-reviewed courses, educational content, recorded courses from CYTO conferences, and a topical webinar series. CYTO U is intended to make cytometry education both accessible and affordable. Students undergoing training at WRHFlow after the 1st of July can choose to become an ISAC member at a discounted student rate that will be included in the training charge. This will provide access to educational material for a year.
For more information on Cyto U click here.
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.
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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, 24th December 2017 until Monday 1st 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.
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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.
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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.
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