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

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 1^st 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 19^th & - October 15^th – 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 19^th 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 19^th 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 4^th – 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 22^nd 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).

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.

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.

1. 41% of the time booked on instruments is not used.

2. 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.