Feb 26, 2026
TBI-ADRD Estrous Monitoring in Mice Standard Operating Procedure
- Kierra Eldridge1,
- Amandine Wislofsky2,
- Gustavo A. Gomez2,
- Andre Obenaus2,
- Paul R. Territo2
- 1Stark Neuroscience Research Institute, Division of Clinical Pharmacology, School of Medicine, IndianaUniversity;
- 2Division of Biomedical Sciences, School of Medicine, University of California, Riverside
- Paul R. Territo: Stark Neuroscience Research Institute, Division of Clinical Pharmacology, School of Medicine, IndianaUniversity;
- TBI ADRD UCR/IU
Protocol Citation: Kierra Eldridge, Amandine Wislofsky, Gustavo A. Gomez, Andre Obenaus, Paul R. Territo 2026. TBI-ADRD Estrous Monitoring in Mice Standard Operating Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbokpqlpk/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: February 20, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 243709
Keywords: Mice, estrous monitoring, vaginal cytology, adrd estrous monitoring in mice standard operating procedure, mice standard operating procedure, tbi adrd closed head injury, estrous monitoring in mice, standard operating procedure, vaginal lavage technique, adrd estrous monitoring, procedure, tbi
Funders Acknowledgements:
NIH NINDS
Grant ID: 1RF1NS138032-01
NIH NINDs
Grant ID: 5R01NS135556-03
NIH NINDS
Grant ID: 7R01NS119605-04
Disclaimer
The authors have nothing to disclose.
Abstract
This standard operating procedure (SOP) document describes the process for performing the common techniques used for estrous monitoring in mice using the Vaginal Lavage Technique. These conditions are standardized to promote reproducibility and are associated with conditions used in the following protocol: "TBI ADRD Closed Head Injury (CHI) Standard Operating Procedure". dx.doi.org/10.17504/protocols.io.14egnrqw6l5d/v1
Attachments
appendix 1.pdf
51KB
Materials
• Pipette (Gilson PIPETMAN 2-20μL Fisher Scientific CAT#F123600G)
• Disposal sterile transfer pipettes (Fisher Scientific CAT# 07-201-927)
• 0.9% Sodium Chloride sterile saline (Henry Schein CAT# 1048583)
• Glass slide (Fisher Scientific CAT# 12-550-A3)
• Coverslip (Fisher Scientific CAT# 12541036)
• 0.1% crystal violet stain (Fisher Scientific CAT# S02514)
• ddH₂O
• Glycerol
• Timer
• Coplin Jar (or other comparable staining vessel)
• KimTech Wipe (Fisher Scientific CAT#6-666A)
• Light Microscope
Troubleshooting
Safety warnings
**NOTE: The tip is placed at the entrance of the vaginal canal and does not penetrate the vaginal orifice. This can avoid injury or cervical stimulation, which can induce pseudo-pregnancy and appear as a persistent diestrus for up to 14 days.
Ethics statement
This protocol follows institutional regulations consistent with the National Institutes of Health Guide for the Careand Use of Laboratory Animals and the ARRIVE guidelines for reporting animal research. Experimental proceduresinvolving animals comply with protocol No. 30608, approved by the Institutional Animal Care and Use Committee(IACUC) at the University of California, Riverside, School of Medicine, and protocol No. 25046 approved by IACUCat Indiana University.
Before start
Scope
The scope of this SOP includes:
- List of materials and required equipment
- Description of the material and the setup of the materials
- Walk investigator through the procedure (with tips where appropriate)
- Analysis of stages
Responsibilities
- SOP Author: responsible for production of the SOP and its described procedures
- SOP Owner: responsible for reviewing and approving changes to the SOP
- Staff: responsible for adhering to SOP guidelines
- NO CHANGES to this SOP are authorized without the explicit approval of the SOP Owner
Introduction
The estrous cycle refers to the reproductive cycle in rodents, and has four phases: proestrus, estrus, metestrus, and diestrus, lasting for a total of 4 to 5 days. The dynamic estrous cycle in which different cell types appear and recede in waves throughout the cycle, reflects changes in the levels of estradiol and progesterone secreted by the ovarian follicles. Assessment of the estrous cycle in experimental animals is a valuable measure of the integrity of the hypothalamic-pituitary-gonadal axis and female fertility. It is essential for experimental design, data interpretation, and reproducibility. It can also be used to investigate the various stages’ effects on the inflammatory process, as well as the impact of drugs, chemicals, and compounds on reproductive function, which can be expressed as a disruption in the typical morphology, cytology, and histology of reproductive organs and an alteration in the duration of each. phase of the. estrous cycle. The cytological evaluation of the 4 stages of the estrous cycle is defined by the presence or absence, and proportions of four basic cell types, as well as by cell density and arrangement on the slide. Vaginal cytology can be evaluated immediately after collection as an unstained wet-mount preparation (direct cytology) or as a fixed, stained slide preparation.
Vaginal lavage-based cytology is a widely used and minimally invasive method for assessing the estrous. cycle in mice. This technique relies on the collection and microscopic evaluation of exfoliated vaginal epithelial cells. When performed correctly, vaginal lavage allows for reliable, repeatable monitoring with minimal stress to the animal. This SOP describes standardized procedures to ensure consistent sample. collection, accurate cytological assessment, and adherence to animal. welfare and institutional ethical guidelines.
Materials & Methods
Vaginal cytology must be collected, evaluated for cycle stage, and documented on the day of experimentation. Despite the common practice of collecting samples early in the morning after the lights are turned on, the procedure can be done at any time of day. To determine the stage of the cycle before or after a particular procedure, the steps should be completed at a single time point (e.g., post-CHI). For CHI experiments, estrous testing will be performed on the day of CHI, but after the mice recover from the CHI.
Load the pipette with 20 µL of ambient temperature sterile saline.
Gently and slowly place the tip into the entrance of the vaginal orifice and flush the liquid into the vagina and draw it back into the tip.
Repeat 5 times to ensure adequate cell retrieval.
**NOTE: The tip is placed at the entrance of the vaginal canal and does not penetrate the vaginal orifice. This can avoid injury or cervical stimulation, which can induce pseudo-pregnancy and appear as a persistent diestrus for up to 14 days.
Document the date and time of the vaginal flush.
Mark the animal (if the group is housed) and return her to the home cage.
After the lavage, the sample is placed on the slide in a thin layer (smear) and allowed to air dry.
Crystals may form on the dry, unstained slides, which will be removed during the staining process.
Dry fixed slides do not require extra fixation before staining.
To stain, place the dry slide in a Coplin jar (or other comparable staining vessel) containing the crystal violet stain and allow to sit for 1 minute; set a timer if needed.
Remove to a second Coplin jar containing ddH2O. Wash the slide with ddH2O for 1 min.
Repeat the staining and washing techniques.
Stain, wash, stain, wash
After you have completed both staining and washing steps, you can remove the excess ddH2O from the edges of the slide with a light-duty tissue wipe, or KimTech wipe, avoiding contact with the stained smear.
Pipette approximately 15µL of glycerol on top of the smear and coverslip.
**Analysis: The stages of the Estrous Cycle are identified by the absence, presence, or proportion of 4 basic cell types, as well as by the cell density and arrangement of the cells on the slide
• Neutrophils
• Small Nucleated Epithelial Cells
• Large Nucleated Epithelial Cells
• Keratinized Anucleated Epithelial Cells
The general approach to staging vaginal smears is to first assess for the presence of neutrophils. If neutrophils are consistently observed, the stage is either metestrus or diestrus; if that cell is absent, it is proestrus or estrus.
Analysis
Proestrus
• Proestrus: This stage lasts around 24 hours in mice. Its main feature is the presence of small, nucleated epithelial cells with relatively uniform appearance and size. Although rare, neutrophils can occasionally be found due to the recent transition from diestrus into estrus. Relatively low numbers of Large Nucleated Epithelial Cells and Keratinized Anucleated Epithelial Cells can be found due to the estrus stage. However, despite the low number of neutrophils or the presence of large, anucleated epithelial cells, this does not compromise the identification of proestrus once the predominant feature of the smear is the small, round epithelial cell population.
Estrus
• Estrus: The estrous stage usually lasts between 12 and 48 hours and is characterized by Keratinized Anucleated Epithelial Cells and bacteria. Nucleated epithelial cells and neutrophils can occasionally appear, depending on the estrous stage (early or late).
Metestrus
• Metestrus: This stage can take between 24 and 48 hours and is characterized by a combination of Keratinized Anucleated Epithelial Cells and Neutrophils. Nucleated cells may appear as well. Since the separation between where metestrus ends and diestrus begins can be tricky, studies display these stages in a condensed form (metestrus-diestrus)
Diestrus
• Diestrus: This is the longest stage of the estrus cycle, with 48 to 72 hours, and is characterized by a substantial decrease of Keratinized Anucleated Epithelial Cells. Overall cellularity includes Neutrophils (in greater numbers) and small and large nucleated epithelial cells.
You can also determine the stage of estrus by visually observing the vaginal opening. This, paired with the vaginal lavage, is the standard practice.
Appearance of the vagina in different phases of estrous cycle of a non-agouti strain mouse. a-Proestrus, b-Estrus, c-Metestrus, d-Diestrus
Protocol references
1. Byers, S.L., et al., Mouse estrous cycle identification tool and images. PLoS One, 2012. 7(4): p. e35538.
2. McLean, A.C., et al., Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification. J Vis Exp, 2012(67): p. e4389.
3. Ajayi, A.F. and R.E. Akhigbe, Staging of the estrous cycle and induction of estrus in experimental rodents: an update. Fertil Res Pract, 2020. 6: p. 5.