Apr 08, 2026
Open field protocol
- 1KU Leuven
Protocol Citation: Ana Cascalho, Nina Schoonvliet 2026. Open field protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxmkylpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 13, 2024
Last Modified: April 09, 2026
Protocol Integer ID: 96557
Keywords: open field protocol, open field protocol this protocol, exploratory activity in rodent, open field, protocol detail, protocol, used assay, exploratory activity, rodent, spontaneous locomotor activity, activity, anxiety, stress
Abstract
This protocol details the Open Field: widely used assay to measure exploratory activity in rodents. It can also be used to infer anxiety, stress and spontaneous locomotor activity.
Attachments
Guidelines
Adapted from:
· Tatem, K., (2014) JoVE & Seibenhener, M., (2015), JoVE
Notes on preparations:
- Prepare open field boxes (50x50cm) and quadrant separators.
- Quadrants should be divided into periphery, non-periphery, center areas.
- Use a room without natural light.
- Mice should be analyzed on the same time of the day consistently with a strong preference for morning.
Materials
- Tripods
- Clean/spare cages
- Timer
- Pens
- Extra cards for animal/cage ID
- LabBook
- Open field arenas
- PC with ANY-maze software connected to a camera
- Ruler + marker if need to remark quadrants
- Spare complete cages with food and water for mice that underwent testing
Before start
Notes on setup:
- If the camera setup is placed high enough, 4 mice can be done simultaneously, BUT make sure the mice do not see each other by placing an opaque separator such as a cardboard.
- Clean and disinfect the cages thoroughly between each animal, no particles should remain between testing sessions/mice.
- Make sure all the quadrants of the open field are properly illuminated. This will facilitate animal detection for quantification afterwards.
- Make sure the video camera catches the full open field and in a straight (non-rotated) manner (this is important for analysis). It is also important that the camera is flat (non-tilted!), otherwise the videos display an angle and are not accurately quantified.
Climatization to testing box (Only needed on unhabituated mice)
10m
Place the mice in the testing room in their home cages for approximately 00:10:00 to acclimate. Leave the room during the acclimation period.
Note
For practicality you can acclimate the mice per cage (e.g. place mice from the same cage together in one open field arena - do not combine mice from different cages!).
10m
Return to the testing room and turn on the activity chambers.
Note
Even though data are not being collected at this time, this will further mimic the testing environment.
Gently remove each mouse from their home cage and immediately place them in the test chambers. If the activity chamber contains a center divider that divides the chamber into quadrants, place one mouse into each empty quadrant.
Once all animals are loaded into the test chambers. Leave the room during this acclimation time.
After 00:10:00 , return to the room and gently return each mouse to his or her respective home cage.
10m
Clean each chamber with disinfectant and paper towels. Ensure no dirt particles are left in the chamber.
Note
- If multiple sessions are being run each day, thoroughly clean each test chamber in between each session with umonium.
- Only perform acclimation prior to the first round of testing to avoid habituation. In addition, randomly assign the animals to a new box each session. Track the box assignments throughout the duration of the study.
Open Field Testing
30m
Place the animals in the testing room for at least 00:30:00 for acclimatization.
30m
Start the computer and open the ANY-maze tracking software (for more information of the ANY-maze software, go to: https://www.any-maze.com/).
Upload your protocol OR make a new protocol.
Define the video sources (4) and apparatus.
Behaviour < Zones < Define the 3 zones: periphery, non-periphery and center.
Periphery: Edge + 6.5 cm
Center: 17.5 cm from the edge.
Behaviour < Immobility detection: Set the immobility sensitivity at 70% and the minimum immobility period as 5s.
Behaviour < Freezing detection: Set the minimum freezing detection as 5000 miliseconds.
Testing < Stages < First stage: fill in the test duration: 1800s.
Go to the experiment tab and fill in the number of animals per treatment that will be tested.
Make sure the mice groups are randomized and run intercalated to avoid hour/group interplay.
Do not manipulate the mice before the test, simply move the mouse from the home cage to the testing chamber and identify the paw tattoo/ear tag after the test is performed.
Move the animal directly to the open field testing box. If you have 4 open field boxes, move each mouse to a box.
Go to the Tests tab and press start all (All 4 mice will be tracked by the software and you can follow it on the screen). Remove yourself from the room or from the mice field of view to avoid interference and unnecessary noises that will interfere with the animal behavior.
When the 30min are finished the software will automatically stop tracking the animal. Move the tested mouse to a new cage if non-tested mice are still present in the original cage.
Clean the testing box and repeat steps 10-14 for the next mice.
Note
- If the mouse is to be tested throughout the study avoid measuring the same animal more than once a month to avoid habituation.
- Randomize order of mice each day to avoid daytime influence.