Can I operate the Agilent Seahorse XF Analyzer myself?

No, at this time we offer Seahorse as a service, whereby SPARC staff help you design your experiment, but we operate the instrument. This protects our XFe96 from damage, and gives you the best quality data as we have a lot of experience setting up these (finicky) experiments.

 

Can users external to SickKids access the Seahorse service?

Yes, as for all SPARC services, we welcome clients outside of SickKids!

 

How does extracellular flux analysis work?

At SPARC, we use an XFe96 Extracellular Flux Analyzer from Agilent Seahorse. The XFe96 measures the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) in a 96-well plate. OCR is an indicator of mitochondrial respiration and ECAR is largely the result of glycolysis. For more information and some excellent videos, please visit Agilent’s website.

 

What is the fee structure for Seahorse XFe96 experiments at SPARC?

Internal SickKids users pay $400 per 96-well experiment, while external users pay $475. This covers instrument time, the Seahorse cartridge plate, the Seahorse microplate for cell seeding, Seahorse media, reagents for a standard Mitochondrial Stress Test (MST) or Glycolysis Stress Test (GST), and SPARC staff time to help design and conduct the experiment, and analyze the data with you.
Note: we do not purchase Seahorse kit reagents but use frozen aliquots for MST/GST experiments. This keeps costs down and has given us reliable, reproducible results thus far. If you would like to use kit reagents, or perform another type of experiment (eg. FAO), you will need to provide the kit and/or reagents.

 

Can I use a portion of the cartridge plate or reuse it?

Unfortunately, the XFe96 is designed to accept a cartridge plate only once, so it’s all or nothing. You can use only a portion of the 96 wells, but the cost of the experiment will be the same, and you can’t use the remaining wells at a later date. Even if the instrument could accept the cartridge twice, the sensors are only good for about 72 hrs post hydration.

 

How do I possibly fill an entire 96-well plate then?

We recommend having 3-6 technical replicates of each condition represented on the same plate; this will improve your data quality and give you better statistical data.

 

Does SPARC provide training for clients?

Partially. SPARC staff will help you design your experiments and teach you how to use Wave software to examine your data. However, there are many informative materials on the Agilent Seahorse website that we encourage you to read or watch, to familiarize yourself with Seahorse technology before you get started. We are not metabolism experts; we’re just getting really good at running these experiments and knowing how to trouble shoot when data doesn’t look good. That is to say, to really understand what’s going on in your samples with respect to metabolism, you will have to educate yourself.

 

What is required then from the user?

You will need to book a date for your experiment, and discuss the type of experiment you wish to conduct with our staff. Once your date is booked, you will be given a microplate on which to seed your cells. Follow this protocol, remembering to keep wells A1, A12, H1, and H12 empty of cells. On the day of your experiment, bring your seeded plate to us, along with a plate map.

 

How do I know what density to seed my cells at?

The Agilent Seahorse website has a fantastic tool (Cell Reference Database – use this link: https://www.agilent.com/cell-reference-database/ ) that provides an easy way to search scientific publications that reference/cite Seahorse XF data. Simply enter your cell type or cell line, hit submit, export the results to Excel, and look for papers that have completed similar Seahorse experiments on 96-well plates. This will give you an idea of what density to start at. However, because everyone’s techniques and cells are different, we recommend that your first experiment includes optimization for cell density. To do this, we typically test 2-4 densities, with 3-6 technical repeats of each.

 

How do I know what concentration of oligomycin or FCCP to use in my assay?

Seahorse used to recommend optimizing for oligomycin concentration in the same way we optimize for cell density; this no longer seems to be the case. From our experience (and likely that of Seahorse), 1 uM oligomycin is almost always ideal. We will be able to tell very quickly from your data if 2 uM oligomycin is needed. However, every cell type seems to require a different concentration of FCCP to achieve the greatest signal. Since you want to know the true maximal respiration of your cells, you want to be working at the optimal FCCP concentration. Too low, and signal will be too low; too high, and signal will still be too low. Thus, we recommend performing a six-point FCCP titration (0.125, 0.25, 0.5, 1.0, 1.5, 2.0) along the x-axis of the plate as part of your first MST experiment. If you truly want to compare max respiration for each different cell line (eg. WT vs. mutant), you may want to titrate FCCP for all cell lines. We realize this can become onerous and expensive – we will do our best to create the most efficient and cost-effective experiments for you. The good news is that most people get usable data even from these initial optimization experiments; think of it as your N=1.

 

What other types of experiments have you conducted on the XFe96?

We have performed customized experiments for those wishing to ask a specific metabolic question, such as “what does the addition of this drug do to the OCR of my cells”? When designing your own assay, keep in mind that any reagents injected during a Seahorse run must be diluted in Seahorse media, so that the instrument truly detects a change in OCR/ECAR and not just a change in solution composition. We have also performed Cell Fuel Flexibility/Preference assays and measured fatty acid oxidation with Seahorse reagents supplied by the user.

 

How do I acquire the Wave software?

The user-friendly Wave software is freely downloadable from the Agilent Seahorse website, as are XF Report Generators (Windows compatible). The software and report generators make preparing Seahorse data for lab meetings and publications very easy and fast!

 

How do I acknowledge SPARC in my presentations or publications?

SPARC normally neither requires nor requests co-authorship on studies using data generated solely on a fee-for-service basis by our facilities. In cases where significant intellectual contributions have been made, co-authorship may be appropriate, following usually-accepted scientific practice.

However, in order to satisfy reporting requirements of our funding partners, and to monitor our scientific impact as a core facility, we require that customers acknowledge work performed by SPARC. This can be done in either or both of the following ways:

1. In the acknowledgments section of peer-reviewed publications, when SPARC personnel have been especially helpful (in study design, data interpretation, or technology selection), and where the author(s) feel that it is warranted, the following sentence should be incorporated:

The authors wish to thank [name of employee(s) and/or director(s)] of SPARC BioCentre Molecular Analysis, The Hospital for Sick Children, Toronto, Canada for assistance with [Seahorse extracellular flux experiments].

2. In the body text of peer-reviewed publications (for example, in Methods and Materials or Results sections), when SPARC has provided a service, the following language should be used:

[Seahorse extracellular flux experiments] were performed by SPARC BioCentre Molecular Analysis, The Hospital for Sick Children, Toronto, Canada.

We will also be pleased to receive notification and a copy when clients publish papers acknowledging SPARC.

 

Can I meet with someone to discuss my Seahorse experiment?

Yes, you can meet with Lilia to discuss your experiment. However, know that we are not experts in your cell type or metabolic question! We are good at designing efficient experiments and can advise you on plate design, reagent concentration, type of experiment, etc, based on our experiences, and can discuss your ideas with you.

 

Who should I contact to book my Seahorse experiment?

Please contact Lilia Baev at lilia.baev@sickkids.ca. We generally respond by the next business day, and typically book experiments a week or two in advance, depending on availability. If you must cancel your booking, as long as you let us know before the cartridge is hydrated (afternoon before experiment day), it’s usually not a problem and you won’t be charged.