July 2019

July marks the introduction of our “application of the month” which will be a reoccurring segment highlighting interesting and relevant applicational notes on particular flow cytometric techniques. Other noteworthy mentions this month concern the MA900, iGrow training, the upcoming MACC meeting, what the Mo-Flo XDP cell sorter can offer you, sheath fluid, and cytokine analysis.

Downtime

All staff operated sorters are unavailable Monday mornings, from 10:00am-11:00am to accommodate a staff meeting. All machines are unavailable on the morning of Tuesday 9th July for a routine deep clean. The Sony’s are unavailable on alternating fortnightly Tuesday mornings for their routine cleans. For up to date instrument availability, please visit our PPMS online booking calendar.

Sony MA900

The MA900 is now live with users having the ability to book the machine with staff assistance. To learn how to operate the machine independently, you can sign up on iGrow. Users who were previously trained on the SH800 can re-attend the training session to be made familiar with changes in both the hardware and software, or can make an assisted booking on the machine and have a member of staff be present to detail these changes in person.

MACC Meeting

Each quarter the Mid Anglian Cytometry Club (MACC) meets to discuss science, trends in cytometry, techniques, and to network with one another.  The next meeting is on Wednesday 17th July 1800-2200 at Sanger (C302).  The program includes talks by Sanger staff; Bee Ling Ng and Annie Speak, and news from the latest Cytometry Conference in Vancouver from Rachael Walker (Babraham) and Mateusz Strzelecki (CRUK). All are welcome to attend.

Beckman Coulter Mo-Flo XDP

The Mo-Flo XDP is a 5 laser cell sorter with up to 15 detectors and a highly customisable optical filter pattern to accommodate complex fluorophore panels. The sorter boasts high recovery, yield, and viability across a multitude of cell types with up to 99% purity within sorted populations. It can sort out of a large array of tube sizes which allows us to sort through large volumes of sample with up to 4 way simultaneous cell sorting to adapt to applications. It can sort into 8-well strips, slides, and plates for both single cell sorting and bulk populations of a cell type which can be cultured after through sterile sorting. It can analyse different light orientations to accommodate unique and niche applications that demand features lacking on other standard flow cytometers. For your next sort session, please make your booking on Mo-Flo XDP.

Flow cytometry sheath fluid

We have nine flow cytometers in the facility, which made use of three different type of sheath fluids.  Sheath fluid is the medium required for focusing the cell suspension into a single stream to be interrogated by the laser though a process known as hydrodynamic focusing.  On the analysers your sample will not come into contact with this fluid until after analysis and on the sorters they will combine with your sample after the laser.  The sorters use autoclaved PBS made by the media team on site, the Cytoflex uses sterile ddH2O, and the BD analysers use the proprietary sheath fluid, BD FACS Flow. PBS, BD FACS Flow and ddH2O can be used interchangeably on analysers with very little effect on data quality.

We are currently investigating moving away from BD FACS Flow for the BD analysers to either PBS or ddH2O.  We want to do this for a number of reasons; shipping 25kg boxes of liquid across countries is not good for the environment, the heavy boxes have been causing health and safety incidents (bad backs), and the benefit of using proprietary sheath is negligible to our users.  Over the next couple of months we will be testing alternatives on our own samples and will let you know the results.  Please contact us if you have any questions or concerns.

Cytokine analysis on flow cytometers

Completing multiple ELISAs can time consuming and can use up large volumes of precious sample.  To get around this you can multiplex your analysis using beads.  The basic idea is that you have a range of barcoded beads specific for each analyte and you do a sandwich ELISA on the beads.  The benefits are reduced sample volume and greater consistency, because you are using the same sample for all of the analytes.  This can be done on a flow cytometer using custom or premade kits:

BD: CBA

Information: https://www.bdj.co.jp/pdf/55-04_04-7900030-3-A1.pdf

Products: https://www.bdbiosciences.com/us/applications/research/bead-based-immunoassays/cba-kits/c/745475

Biolegend: LEGENDplex

Information and products: https://www.biolegend.com/legendplex

ELISAGenie: GeniePlex

Information and products: https://www.elisagenie.com/genieplex

Please contact us for advice if needed.

 

Application of the month: Apoptosis measurement

This is a method of distinguishing self-induced cell death from other causes of necrosis. Using Annexin V and PI provides the opportunity for researchers to investigate and comparably measure the difference in these different pathways for cellular death which can prove useful later for downstream applications and data analysis.

Using a live/dead stain in your experiment is essential to exclude cells that may exhibit unusual staining and are not the cells you want to analyse.  These stains only detect dead cells, not cells that have begun the cascade towards cell death.  This is why checking for apoptosis is useful prior to sorting or data analysis, as these cells may adversely affect your cell culture (release of cytokines, DAMPs, etc.) or display unusual staining patterns.

Phosphatidylserine is a phospholipid and component of the plasma membrane located on the inner leaflet of the cell. During apoptosis the plasma membrane is depolarised which inverts phosphatidylserine to the outer leaflet as a signal for the cell to be phagocytised. Annexin V is a cellular protein that binds to phosphatidylserine which can be conjugated to a fluorophore to determine the level of apoptosis within a sample population. Used in tandem with a simple live/dead stain such as Propidium Iodide, which binds to the DNA of cells which have compromised and permeable membranes, creates an assay to measure and compare levels of apoptosis and necrosis.