CytoFLEX Cell Counter / Flow Cytometer to be installed Tuesday 30^th 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.

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 1^st 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 21^st of December as they will be shut down for the Christmas Closedown. The sorters will also not be open for bookings on Wednesday the 3^rd 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 will become 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.

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

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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.

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Levitas LeviCell Trial – ITS HERE NOW – February 22^nd to March 5^th 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.

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Unused Analyser Session Charges

Starting on the 1^st 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.

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Westmead Cytometry User Group Meetings

‘2^nd 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.

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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 11^th 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.