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