MethodologyHistory of Facility The Purchase

Assessing Needs

The Future...

Meeting the Needs of our Customers: Assessing Sorters to grow our Flow Cytometry FacilityIsobel Darlington, Aleksandra Lazowska, Attila Bebes, Rebecca Roberts, Arthur Davis and Rachael Walker

Flow Cytometry Core Facility, Babraham Institute, Babraham Research Campus, Cambridge CB22 3ATwww.babraham.ac.uk@babrahaminst

Flow Cytometers have been used forresearch at Babraham Institute (BI) sincethe mid-1980’s, with the Flow CytometryCore Facility being established in the mid-1990’s. It’s primary aim is ‘to provideflow cytometry service to enhance andadvance Institute science, utilising state-of-the-art equipment’.

Advancement in technology along withthe need for more sophisticatedequipment to answer BI scientist’shypotheses, has meant that BI hasremained and in some cases been aheadof the trends in cytometry equipment.

FACS 420 machine @BI in 1987 was run by flow cytometry specialist

Nigel Miller

The main BI users of the Facility are the Immunology groups, who require multi-parameter cytometers for both analysis and cell sorting. The Facility is equipped withhigh-end analysers such as 3 x BD LSRFortessa, a Propel Labs YETI, an AmnisImagestream and our latest addition is a Cytek Aurora Spectral Analyser. To help paneldesign and planning sorter configurations (FACSAria, FACSAriaFusion, Influx) are matchedas close as possible to the analysers.In recent years there has been a huge growth in the amount of work that the facilitycarries out for Biotech companies in and around Cambridge, especially those housed atthe Babraham Research Campus (BRC).

During the past 5 years, the Facility has seen an increase in the number of requests for aFlow Cytometry service from Biotech companies based on BRC and around theCambridge area (between 2014 – 2019 the hours of sorting carried out for biotechcompanies increased ~180%). We have managed this by using any unallocated capacityon the BI sorters for external work, which has generated income to allow the Facility staffnumbers to expand. However, as priority is given to internal scientists, we often did nothave capacity to accommodate external work.

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Hours sorted based on number of fluorochromes

FP Multi Total

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Hours of Sorting Carried out in Facility split by User Type

Institute Campus Company External Company

The demand for large-scale library sorts of CRISPR edited cells (using GFP or RFP) bycompanies has increased significantly over the last two years (>30% of sorts carried out2016- 2019 were for GFP/RFP).

Graph showing total hours of sorting for all users also broken down into those using only 2 or 3 colours

(GFP/RFP/Viability) called FP and those with more fluorochromes (multi)

Graph showing total hours of sorting split into users type. Institute users, those based on BR Campus

companies and external companies.

In order to meet this demand, it was decided to invest in additional instruments thatwould allow efficient sorting of large numbers of cells using small panels (2-3 fluorescentparameters). Any new pieces of equipment need to be cost-effective and generateincome for the facility. A series of tests were carried out on sorters including, speed,efficiency, purity and yield of sorts, as well as single cell deposition to ensure accuracy. Inaccordance with Institute Biosafety Regulations all new sorters had to be housed in aBSL2 cabinet.

During our years of sorting we have always found that the Jet in Air systems (Influx andMoFlo) are quicker for sorting than the cuvette based systems (FACSAria andFACSAriaFusion). This has meant that we have always placed screening sorts whichrequire bulk sorting of hundreds of millions of cells onto our Jet in Air systems.Therefore, it was a prerequisite to acquire an additional Jet in air system for the Facility.

Bead Based TestsWith any new sorter within the Facility, wealways carry out a series of tests to ensurethat the machine has good resolution of 8peak beads. We also test the depositionof a single FlowCheck Bead (BeckmanCoulter) per well of a 96/384 well plate.The wells studied microscopically toensure that each well contains a singlebead. We have also in the past sortedindividual cells and looked for GAPDHhousekeeping gene to ensure that a cellwas present in a well, however this wasnot carried out during this study. Comparison of 3 sorters with deposition of FlowCheck

beads into the wells of a 384 well plate. Results show the FACSJazz has the best results.

Cell Based testsWe are often asked to sort cell lines such as HeLa and HEK 293 for companies which havebeen transfected or Gene Edited. For these cells we set up our sorters with a 100 µmnozzle (Influx at 30 PSI) and we run the cells at around 5000 events per second. Theselarge cells however, disrupt the sorting streams so we have to pause the sorts frequentlyto clean the plates.

For our tests, we tried 2 commercially available jet in air systems (both of which aremarketed for use for GFP/RFP sorts due to their ease of use and reduced number oflasers and detectors). Multiple sorts were carried out on the same cell types comparingthe sorts, looking at time taken to sort, efficiency, purity and viability on these twomachines as well as the Facility’s Influx sorter.

Following our tests we found that each of the sorters gave similar results to our Influxsorter. However, we decided that deposition into a 96/384 well plate was important. Wehad the opportunity to purchase two second-hand BD FACSJazz sorters.

We decided to purchase these two sorters housed in Baker-Ruskin BSL2 Hoods in EarlySpring 2019. By running two sorters in parallel for these library screens, this allows thecompany to sort more cells faster and they can generate a larger library. This is alsoimportant as it means that we can carry out the company work quicker and this thenfrees up more time on the other sorters for Institute work.

The purchase of these sorters has had several other positive effects on the Facility:• Having this service for efficient bulk sorting for CRISPR edited cells, has subsequently

been used by more companies and Institute users.• The investment into these sorters, has increased the number of hours of sorts being

carried out and increased the income into the facility (see graph below)• By moving all ‘simple’ GFP/RFP sorts to the Jazz sorters, this has freed up time on the

high-end sorters for more complex sorting.• Having extra sorters, has allowed us to expand our services to include Yeast and

Bacteria sorting which were previously not permitted on our sorters.• As the two ‘new’ sorters are in a separate room to our other sorters, this has allowed

more sorting to be carried out during the COVID-19 pandemic.

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Hours of sorting in BI Flow Facility broken down by machine(Apr-19 - Mar 20 *)

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* = lockdown enforced by UK government on 21st March. Allexternal sorts cancelled and only Critical BI sorts carried out

Graph showing hours of sorts carried out in past year. The use of the Jazz machines is sporadic due to company projects but the breakdown shows the

machines are heavily used.

The BI Flow Cytometry Facility continues to look at the Cytometrymarket for new and exciting pieces of equipment to complementand advance the technologies we already offer. It became apparentthat it is crucial to have sorters with a range of capabilities within adynamic Flow Cytometry Core Facility.The Facility continues to assess and test new sorters on the marketto see if they meet the needs of both the multi-parameter Institutescience with the speed for bulk sorting for Biotech companies.We are especially keen to evaluate new Jet In Air sorters as we see

these as a key piece of technology for our core.

Other things that were taken intoconsideration were:• Ease of use• Being able to sort into 96/384

well plates• Cost of sorter• Size of sorter and size of the

BSL2 hood.

Data showing cells sorted based on GFP low/high expression. Cells sorted on Enrich mode.