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CytoFLEX Cell Counter / Flow Cytometer to be installed 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.

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

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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' 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

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!

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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.

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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?

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The UV laser has changed on the Fortessa

See here for continuing use of application settings

Student training gets a boost - Cyto U access

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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, 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.

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