Moving on..

As the year comes to a close, so does my additions to the Garvan Flow Cytometry Blogger and Garvan Flow Twitter feed (twitter.com/garvanflow)

The new year brings exciting changes and interesting times, and in light of this I have decided to centralise my web content via a new Blogger simply called Chris Brownlee which can be found at http://tiny.cc/oezjo or below

Thank you all for reading and I look forward to continue spreading the work we do via the following means

words via Blogger: 

http://www.blogger.com/profile12158037879752405544

posts via Twitter

http://twitter.com/#!/UNSWFlow

and photos via Instagram: 

chrisbrownlee

I wish you all a very Merry Christmas a Happy and productive New Year!

Best wishes,

Chris

BD FACS Jazz™ Install

New gear and the New year..

So, its been a while since I added anything to this blog and thought it about time to fill you all in on the facility changes, my new job and the continuing awesomeness of the MLC Community Foundation Flow Cytometry Facility at the Garvan Institute

First up, the new guy. We were fortunate enough to have one exceptional applicant in our latest round of offers looking for our newest Flow Cytometrist in the community, Mr David Snowden. David joined us earlier this year from UTS where completed his degree in Forensic Science. With all aims to continue studying, and being accepted into a Masters of Biomedical Engineering, David saw the light, put the Masters on hold, and focused on the job at hand and completely immerse himself in the Flow world. In the last few months David has excelled in his performance as an operator and is showing true signs of commitment to the field and we are super lucky to have him at the Garvan, and in the industry as a whole. Thanks for everything mate and all the best in the years to come. Special thanks to Nikki for doing such a smashing job on not only keeping up with the huge number of bookings and sorts we do every week, but to find the time to train, support and teach David. I definitely could not have done the last 2 years with out you..this post is starting to sound suspiciously like a farewell..

Secondly, the new gear. BD FACS Jazz™. Brent, UC Flow and pretty much everyone that has worked on one of these little bad boys was right..we LOVE it!! Amazing little Flow Cytometer and perhaps the best thing on the market at the moment. We are blown away by its size, accuracy, modularity, design, software, speed and purity, its a screamer! 2 way sorting off 8 parameters at its absolute best, here are some pics of the install to keep you up to speed should you ever be fortunate enough to install one in  your lab.

Crate arrives, out come the crowbars

The BD FACS Jazz™

The trolley of accessories! Christmas came early!

2 men easily lift it on the bench

Approved and in place

Organising a table for the electronic components

FSC, SSC, 4 off the blue and 2 off the red

640nm comes installed and mounted on an angle for polarisation

Computers hookup

Pressure is digitally regulated, likewise sample offset is linked to sheath pressure

488nm is mounted, practically aligned by itself. 

Brent - the machining and design of this is incredible as usual - thanks so much!

New BD service engineer inspects the optics

Computers are hooked, software is launched

A very familiar nozzle assembly starts to take shape

Front view of instrument

Digitally processed images of the familiar Influx windows

488nm and 640nm hit the scatter bar

Influx and Jazz side by side in the lab

The workstation - cosy!

Red and blue tweaking

8 peak Rainbows on the R660 and R780 channels on BD FACS Jazz™

8 peak Rainbows on the R660 and R780 channels on BD Influx™

There you have it, installed and ready to go.

We 2-way sorted the second day post install and we are most impressed. I had the entire side panel off and was in looking at the electronics, super compact! I discovered that there may be some room to move on modifications with this instrument, after all.. it is an Influx right?

Thanks so much BD, Cytopeia, Brent, Ger, all the team in Seattle, all the guys in San Jose, Darren, Adam, Christine, Marlene, Andrew, all the team and most importantly Sladjan for always getting us up an running and most of all thanks to the Garvan Board of Directors for funding us! John and Cherry, you are truly amazing. I have not once received a 'no' from either of you and I can't thank you enough for that.

I am signing off the garvanflow blogspot and twitter feed and will commence again under a new banner when I take up my new position as Flow Cytometry Scientist at The Mark Wainwright Analytical Centre of University of New South Wales on January 3rd. David will keep up the posts and keep you all informed of the upgrades, changes and happenings here in the MLC Flow Cytometry Facility at the Garvan Institute.

Finally, I would like to welcome Mr Robert Salomon and wish him all the best in his new role as Manager, Flow Cytometry. Congrats mate and all the best for the year ahead

Until next time, yours truly

Chris Brownlee

BDInflux™ is up and running

Its been 7 weeks since I got back from Cytopeia and I have done little other than run Influx since I got back, the hours spent on the instrument are already too many to count and its paying off in unimaginable ways. We got over 80% efficiency in single cell sorting last week, one cell per well, with 3 from 16 cells coming up with positive bands after PCR. On repeat with the next 16 wells a further 4 came up positive. An impressive result from a sort that was set up after 5 hours battling a failed sheath pressure transducer on FACSAriaIIu only to bail once we realised the plate wasn't able to respond in the Y-direction (later to find the serial port had not been plugged back in after a sample agitation motor was replaced - but thats another story). So, 5 hours down, almost 12 hours in the day with half an hour lunch, the Influx was tackled head on with a 4 colour, 4 laser sort and did it ever shine. We had some problems with drop charge contacts and tracked the problem down using scope probes, multimeters, plate voltage read outs, and after replacing the contacts on the shoe behind the nozzle assembly a second time (this is why it was overlooked - twice was totally unexpected) we have now got some seriously reliable side streams. What I have benefited from most here is the knowledge of changing sort driver racks, trouble shooting over the phone with Brent, learning about software, getting the builds installed and updated, replacing high voltage boards etc. It has been a thorough learning experience and I can now say the setups are taking me an hour, the experiments are taking an hour to set up and the sort button is ready to hit after 2 hours, 11 colour panels, 6 way sorting. Sweet. Its been nothing less than epic but feel we are well on the way to some amazing sorts in the near future, first up being cell cycle sorting and microparticles. Also, if anyone has ideas about esophageal cell markers I would be most interested.

So thats me for April, will keep you updated as things keep progressing. Thanks BD for the opportunity to talk in Adelaide, I look forward to the next one.

©

inFlux Training at Cytopeia

I just wanted to post to keep things consistent with our installation of the 7-laser inFlux at Garvan as I have just completed a weeks training with Brent from Cytopeia BD in Seattle and it has been nothing short of amazing and absolutely invaluable.

The week consisted of laser installation, alignment, optimisations, changes, modifications, new technology, advancements to current equipment, systems and so much more. I know feel closer with my instrument, ready to use it for everything it was designed for and have the confidence to take it to the levels of research that are yet to be defined. The inFlux truly is that instrument. In true Ger style, nothing is impossible.

It was an amazing week. We enjoyed lunch with Andrew at the cuban sandwich bar, a beautiful dinner with Brent, Ger and his wife Barb (thank you both so much). Day trips, home cooked meals, family, friends and science. What ever could be better! I have been blessed and truly spoiled by the wonderful hospitality, incredibly intelligent people I have met, all who are innovative, creative, wonderful and fun.

Thank you to all at Cytopeia. Thank you to BD for giving me this opportunity and most of all, thank you Brent for being a fantastic host, I am looking forward to hanging out down under when your next in town.

I hope these messages help users understand the outcomes of investing in BD in particular the inFlux technology, its an amazing time in research and when you are fortunate enough to purchase equipment like this you truly are getting so much more than just an instrument. My goal is to share these experiences with users in order to create interest, help the companies grow and together, expand and develop what I believe to be the future of scientific research in medical advancements for all humans and the greater good of flow cytometry and even science as a whole.

Best wishes,
Chris

Influx Training

Friday 00:01am

Influx Training completes today, its been a fantastic first week of  2011 back at work. Darren Ellemore has been a fantastic support, an abundance of knowledge and a great trainer, his attention to detail and meticulous procedures help significantly when getting used to this instrument. Overall, the week has been very enjoyable and we have learned alot. Tomorrow is our last day but we feel the support will always continue.

I feel I should comment on thoughts about the instrument, and I don't want to repeat myself at all so I will just say, not only has this instrument given us the ability to get through more samples from users and opened up an entirely new spectrum of experiments but it's also given us a clearer knowledge and fundamental understanding about flow cytometry. Aside from its open architecture that may deter some core operators, its by far the cleanest, easiest, simple yet complex instrument in our fleet.

Darren and BD, Thank you!

Chris

Day 4 - BDInflux™ Installation

Day 4 - Influx installation complete!

Again, I got in early to repeat the start up procedure and, as it should, she started up well and lasers required minimal alignment even with a tip change. This truly is the easiest instrument in the BD range to set-up, align and even teach others about Flow Cytometry (providing you have great engineers setting you up of course). Nikki with only 8 months of Flow under her belt has found the instrument to be very easy to setup and spent some time swapping nozzles over, moving the stream about and still has very little to do alignment wise. Its a beautiful thing aligning a stream to fixed objectives rather than the other way around (Vantage) it makes so much sense and is proving to be a joy to start up in the mornings.

After a morning of trouble shooting the 6 way separation, we made a few slight adjustments to the deflection plates and saw that the 'bowing' affect on the 6 streams was reduced significantly thanks to Brent's modifications, so, we were all set to show off our new instrument and skills to visitors later that day.
Centenary Institute's Dr Adrian Smith and Robert Salomon arrived in the early afternoon and we showed  sorting beads using the 6 way collection tubes, they were also interested in changing PMT detector blocks and some features of the Influx like sorting on Time as a parameter and also Index sorting. Everything went according to plan (or course) and they left satisfied. BD were great in allowing me to show them the instrument and I really appreciated Darren's offer to do the demo. Brent was there to support the higher end questions that were asked and together I feel we pulled it off. Later that afternoon, I began to shutdown so I started to pack up and head home. A pat on the back from Mark and a comment about running on fumes sealed the deal and I headed off.

Brent - I can't thank you enough for everything you have done for us with the build of the instrument, the quality of the installation was of extremely high standards and you have provided us with an amazing platform for our researchers to utilise. Mark - you have been a rock this week for us and your help, guidance, information and conversations about the instrument have been put so simply that I feel you have given us the confidence we need to really run with this so thank you so much. Darren, your knowledge of the instrument is so thorough, your experience with sorting from your old days, your a wealth of information and I am really looking forward to our training that begins in a couple of weeks so thank you and we are keen to run some panels and get used to the workflow of FACS Sortware™. Craig, having you here has been a great addition and I hope you have enjoyed your time in Sydney, I look forward to bouncing ideas with you and calling on your expertise from time to time, thank you. Also, thanks to all at BD especially Christine Bligh & Marlene Daalmeyer for their help, support, professionalism and planning to get us to this point. Thank you. Adam your help and mate-ship is much appreciated as always, we'll see you soon for a demo.

Finally to Ger, thank you, again, your ideas and expertise behind building the Influx are so greatly appreciated, from someone like myself and Nikki that run Vantage everyday, we really see the differences you have made to Flow Cytometry as a whole with this instrument and I can't thank you enough for such a beautiful piece of engineering brilliance. I look forward to seeing you soon in Seattle for Advanced hardware training with Brent (an amazing opportunity that I am very grateful for).

I would also like to thank Dr Stuart Tangye and Dr Robert Brink for the opportunity to run such an amazing instrument in such an incredible institute, our facility is proving to be a real hub for research and I am so grateful to be a part of it all. Thank you both so much

On that note, I will sign off from the Influx installation blogs, and until the LSR7 is installed, stay tuned. Thanks for reading.

(from left back row clockwise, Craig Kelsey, Sladjan Milanovic, Mark Yoneyama, Brent Harrison, Darren Ellemore, Nikki Alling and me)

Best wishes,

Chris

ps, Marlene, we named the instrument the DMC-12 after the beloved DeLorean car from the Back To The Future movies - with its Flux capacitor how could we resist.

Day 3 - BD Influx™ Installation

Day 3 BD Influx™ Installation; Software, Six way Cell Sorting and Celebrations                       

Day 3 began again early, in an attempt to get some ‘me –time’ with the instrument I arrived at a silly 7am again, changed the compressor over to house air, turned the laser keys one – by – one and fired her up. After some minor sheath tank troubleshooting the pressure gauge soon read a steady 60psi and I began playing around with the stream. It wasn’t long before I ran into trouble and my saviour Darren walked through the lab doors dot-on 8am.

“Questions” was my first word (after good morning of course) so, we began the correct stream start up procedures and away we went, off on the 86um tip again we aligned the instrument a 488nm first, and began the process of fine-tuning each consecutive laser. Thanks to the scope, the timing of each laser is incredibly simple to get your head around, each pulse peaks in 2 channels depending on which PMT’s your interested in (simple click on a dot-plot will select which 2 channels to look at) X and Y come up on the scope, and underneath you have buckets where each pulse is to be detected, by moving the timing of the laser in the software the buckets shift left or right, align a bucket under a pulse and bingo you have a signal. Amazingly, the majority of the alignment is done by looking at beams through pinholes on the pinhole camera, focal points within those pinholes help to peak the signals, its only at each final point that we sweep a laser and watch the signal on the digital acquisition templates. Once I get a few more hours on her I know this will become very quick.

I change my first filter and move my first detector block out of the way to do so, simply, it works and no light is ever able to enter the casing that contains the filters, we install the 70um and amend our settings (the usual suspects; timing delay, sheath pressure and frequency). The cells are due to arrive at 3pm and we spend an hour ensuring each stream hits the 6 collection tube perfectly (all is done by moving the stage), samples are run and single colour controls are acquired, gates are set and we steadily chip away at the Sortware™ gating positive/negative population and dropping them into the compensation layout matrix. It has a FlowJo feel and the matrix is set smoothly and quickly, overlaps are minimal (except for a few controls that didn’t work – which ended up creating 117% overlap, which happens) and we soon begin sorting our populations. We both sigh as the sort button is clicked and purity checks reveal that all 6 were successfully collected. Workflow is still a little clumsy as get where we need to be, but perhaps not in the most effective way but are soon rewarded with dots in the respective gates on purity check.

As we head to dinner, the sun sets behind the cityscape and we sip Italian beer while feasting on carpaccio, zuchinni flowers, sorbet pallet cleansers pork bellies and fish-in-a-bag. Crème brulee and profiterolls see us waddle out at 10:30pm and I jump a train home.

First day setting up from scratch and sorting 6 populations J I am amazed at the speed in which this has all been possible. Joe, the software is perfect, Ger your instrument is elegant, rich and complex yet simple, seamless and solid. Thank you BD!! day 3 was amazing.

Chris