Apr 28, 2026
Three-Chamber Social Preference Test in Mice
- Sarah van Veen1,2,
- Zsuzsanna Callaerts-Végh1
- 1KU Leuven;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Sarah van Veen, Zsuzsanna Callaerts-Végh 2026. Three-Chamber Social Preference Test in Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrbnqolmk/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: April 21, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 315434
Keywords: ASAPCRN, chamber social preference test in mice, chamber social preference test, mice this protocol, social preference test, behavior in adult mice, social preference trial, adult mice, mice, social novelty trial, maze tracking software, habituation trial, behavior, sniffing zone, chamber, central chamber, linear transparent plexiglass apparatus, protocol
Funders Acknowledgements:
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Abstract
This protocol describes the three-chamber social preference test to assess pro-social behavior in adult mice. The setup consists of a linear transparent plexiglass apparatus with a central chamber connected to two side chambers via perforated walls. The protocol includes a habituation trial, a social preference trial (Stranger 1 vs empty), and a social novelty trial (Stranger 1 vs Stranger 2). Time in sniffing zones is recorded using an overhead webcam and ANY-maze tracking software (Stoelting Europe, Ireland).
Guidelines
• Stranger mice must come from different home cages and be unfamiliar to the test mouse.
• Stranger mice should be sex-matched and age-matched to the test mouse.
• Counterbalance the side (left/right) of the stranger mouse across animals to prevent side bias.
• Clean the setup with 50% ethanol between animals to remove odor traces.
• Test should be conducted at consistent times of day under dim lighting.
• Up to 4 animals can be tested in parallel.
Materials
| A | B | |
| Subjects | Adult test mice (WT/KO, ~2 months old) + unfamiliar stranger mice (same sex, same age) | |
| Apparatus | Three-chamber box: central chamber (40 × 10 cm) + two side chambers (10 × 10 cm each) | |
| Chamber divider | Perforated wall (0.8 cm diameter holes) allowing olfactory/visual/auditory but limited tactile contact | |
| Sniffing zones | 5 cm defined zones in front of each side chamber | |
| Recording | Overhead webcam + ANY-maze tracking software (Stoelting Europe, Ireland) | |
| Cleaning | 50% ethanol + absorbent wipes |
Before start
• Confirm stranger mice are from different home cages, sex-matched, and age-matched to test mice.
• Counterbalance left/right placement of Stranger 1 across animals.
• Calibrate ANY-maze: define central chamber, two side chambers, and sniffing zones in front of each side chamber.
• Allow test mice to acclimate to the procedure room for 30 min before testing.
• Prepare 50% ethanol and wipes for thorough cleaning between animals.
• Set up enclosure with dim lighting and confirm overhead webcam is recording correctly.
Habituation trial (5 min)
Place the test mouse in the central chamber of the empty apparatus. The side chambers are not
accessible during this phase, the test mouse is confined to the central chamber only. Record
locomotion for 5 min. This habituates the animal to the novel environment.
Note
Do not allow the test mouse to access the side chambers during habituation.
Social preference trial (10 min)
Without handling the test mouse, gently introduce an unfamiliar stranger mouse (Stranger 1, S1)
into one of the two side chambers. The opposing side chamber remains empty (E). The position of
S1 (left or right) is randomized across animals to prevent side bias. Open access to all three
chambers and allow free exploration for 10 min. ANY-maze records time spent in the sniffing zone
adjacent to S1 and in the sniffing zone adjacent to the empty chamber.
Social novelty trial (10 min)
Without removing the test mouse, introduce a second unfamiliar stranger mouse (Stranger 2, S2)
into the previously empty side chamber. Allow free exploration for 10 min. ANY-maze records time
spent in the sniffing zone adjacent to S1 (now familiar) and in the sniffing zone adjacent to S2 (novel
stranger). A preference for S2 indicates intact social novelty recognition.
Return test mouse and clean
After the final trial, return the test mouse to its home cage. Remove stranger mice. Thoroughly clean
all chambers, walls, and floor with 50% ethanol. Allow to fully evaporate before the next test animal.
Data analysis
For the social preference trial: calculate time in S1 sniffing zone vs E sniffing zone per animal.
Social preference index: (S1 zone time) / (S1 + E zone time). For the social novelty trial: calculate
time in S2 sniffing zone vs S1 sniffing zone. Compare indices and raw zone times between
genotypes using appropriate statistical tests.
Expected result
WT mice typically spend significantly more time in the S1 sniffing zone than the empty zone during the social preference trial, and more time with S2 than S1 during the social novelty trial. Reduced preference in KO animals suggests impaired social motivation or novelty recognition.