Essential 10

9. Experimental procedures What was done, how it was done, and what was used. explanation

For each experimental group, including controls, describe the procedures in enough detail to allow others to replicate them, including:

9a What was done, how it was done, and what was used.
9b When and how often.
9c Where (including detail of any acclimatisation periods).
9d Why (provide rationale for procedures).
Explanation
Examples

Essential information to describe in the manuscript includes the procedures used to develop the model (e.g. induction of the pathology), the procedures used to measure the outcomes, and pre- and post-experimental procedures, including animal handling, welfare monitoring and euthanasia. Animal handling can be a source of stress and the specific method used (e.g. mice picked up by tail or in cupped hands) can affect research outcomes [1-3]. Details about animal care and monitoring intrinsic to the procedure are discussed in further detail in item 16 – Animal care and monitoring. Provide enough detail to enable others to replicate the methods and highlight any quality assurance and quality control used [4,5]. A schematic of the experimental procedures with a timeline can give a clear overview of how the study was conducted. Information relevant to distinct types of interventions and resources are described below.

Examples of information to include when reporting specific types of experimental procedures and resources
Procedures Resources

Pharmacological procedures (intervention and control)

  • Drug formulation
  • Dose
  • Volume
  • Concentration
  • Site and route of administration
  • Frequency of administration
  • Vehicle or carrier solution formulation and volume
  • Any evidence that the pharmacological agent used reaches the target tissue

Surgical procedures (including sham surgery)

  • Description of the surgical procedure
  • Anaesthetic used (including dose and other information listed in pharmacological procedures section above)
  • Pre and post analgesia regimen
  • Pre-surgery procedures (e.g. fasting)
  • Aseptic techniques
  • Monitoring (e.g. assessment of surgical anaesthetic plane)
  • Whether the procedure is terminal or not
  • Post-surgery procedures
  • Duration of the procedure and duration of anaesthesia
  • Physical variables measured

Pathogen infection (intervention and control)

  • Infectious agent
  • Dose load
  • Vehicle or carrier solution formulation and volume
  • Site and route of infection
  • Timing or frequency of infection

Euthanasia

  • Method of euthanasia, including the humane standards the method complies with, such as the American Veterinary Medical Association (AVMA) [6]
  • Pharmacological agent, if used (including dose and information listed in pharmacological procedures section above)
  • Any measures taken to reduce pain and distress before or during euthanasia
  • Timing of euthanasia
  • Tissues collected post-euthanasia and timing of collection

Cell lines

  • Identification
  • Provenance
  • Verification and authentication
  • RRID [7,8]

Reagents (e.g. antibodies, chemicals)

  • Manufacturer
  • Supplier
  • Catalogue number
  • Lot number (if applicable)
  • Purity of the drug (if applicable)
  • RRID

Equipment and software

  • Manufacturer
  • Supplier
  • Model/version number
  • Calibration procedures (if applicable)
  • RRID

Where available, cite the Research Resource Identifier (RRID) for reagents and tools used [7,8]. RRIDs are unique and stable, allowing unambiguous identification of reagents or tools used in a study, aiding other researchers to replicate the methods.

Detailed step-by-step procedures can also be saved and shared online, for example using Protocols.io [9], which assigns a Digital Object Identifier (DOI) to the protocol and allows cross-referencing between protocols and publications.

 

References

  1. Clarkson JM, Dwyer DM, Flecknell PA, Leach MC and Rowe C (2018). Handling method alters the hedonic value of reward in laboratory mice. Scientific reports. doi: 10.1038/s41598-018-20716-3
  2. Gouveia K and Hurst JL (2017). Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling. Scientific reports. doi: 10.1038/srep44999
  3. Hurst JL and West RS (2010). Taming anxiety in laboratory mice. Nat Methods. doi: 10.1038/nmeth.1500
  4. Hewitt JA, Brown LL, Murphy SJ, Grieder F and Silberberg SD (2017). Accelerating Biomedical Discoveries through Rigor and Transparency. ILAR journal. doi: 10.1093/ilar/ilx011
  5. Almeida JL, Cole KD and Plant AL (2016). Standards for Cell Line Authentication and Beyond. PLoS Biol. doi: 10.1371/journal.pbio.1002476
  6. Leary SL, Underwood W, Anthony R, Cartner S, Corey D, Grandin T, Greenacre C, Gwaltney-Bran S, McCrackin M, Meyer R, Miller D, Shearer J, Yanong R, Golab G and Patterson-Kane E (2013). AVMA guidelines for the euthanasia of animals: 2013 edition. https://www.avma.org/KB/Policies/Pages/Euthanasia-Guidelines.aspx
  7. Bandrowski AE and Martone ME (2016). RRIDs: A Simple Step toward Improving Reproducibility through Rigor and Transparency of Experimental Methods. Neuron. doi: 10.1016/j.neuron.2016.04.030
  8. Bandrowski A, Brush M, Grethe JS, Haendel MA, Kennedy DN, Hill S, Hof PR, Martone ME, Pols M, Tan SC, Washington N, Zudilova-Seinstra E and Vasilevsky N (2016). The Resource Identification Initiative: A Cultural Shift in Publishing. J Comp Neurol. doi: 10.1002/cne.23913
  9. Teytelman L and Stoliartchouk A (2016). Protocols.io: Reducing the knowledge that perishes because we do not publish it. Information Services & Use. doi: 10.1371/journal.pbio.100253