Murine Nasal Lavage Fluid Collection without Blood Contamination

Our research focuses on developing a method to collect nasal lavage fluid from mice in large volumes, addressing two main challenges: achieving high yield and preventing blood contamination.

There are two approaches to collecting nasal lavage fluid: the pharyngeal route, which yields larger volumes, and the tracheal route, which reduces blood contamination.

Our method enables the collection of large volume of nasal lavage fluid from the pharynx, while placing cotton balls in the rodent's mouth helps minimize blood contamination. The approach is both simple and cost-effective.

To detect blood contamination in the sample, we employ the forensic method, known as a luminol reaction. The luminol reaction is a simple and more sensitive than other methods.

The concentration of IgG in blood is much higher than that in nasal lavage fluid. Our method, which avoid blood contamination, is crucial in immunological research. It is also suitable for analyzing other molecules, such as the microbiome, where blood contamination can compromise accuracy.

[Narrator] To begin, place the euthanized mouse on its back on a surgical plate, and fix all four limbs using pins. Using a one-milliliter syringe, collect blood from the heart. Using scissors, open the abdominal cavity to expose the visceral organs and diaphragm. Incise the diaphragm and ribs to reveal the right atrium. Then, puncture the right atrium with scissors to drain remaining blood. Place three cotton balls into the mouth of the animal while pulling the tongue forward. Then, raise the head into a nose-up position, and use scissors to separate the lower jaw to prevent blood contamination in the nasal lavage fluid. Replace the cotton balls with fresh ones to avoid blood contamination. After bleeding has stopped, inject 200 microliters of sterile PBS into the choana and collect the nasal lavage fluid ejected from the nostrils into a 1.5-milliliter tube. To prepare the stock solution for chemiluminescence detection, dissolve one milligram of luminol and five milligrams of sodium peroxide in one milliliter of sterile water inside a 1.5-milliliter tube. Cover the tube with aluminum foil, and store it at four degrees Celsius until use. Next, prepare a working solution by diluting the stock solution tenfold with deionized water. Pipette two microliters of the nasal lavage fluid sample into wells of a 96-well plate. Add 100 microliters of the diluted luminol solution to each well, and briefly shake the 96-well plate to mix the contents. Directly observe the chemiluminescence reaction in the dark. This figure presents a comparison of immunoglobulin A concentrations across different sample types. In nasal lavage fluid, the concentration of immunoglobulin A is lower in samples collected using the novel method compared to the conventional method, whereas serum and bronchoalveolar lavage fluid samples show no substantial differences between the two methods. This data suggests that the novel method reduces contamination in nasal samples, leading to more accurate immunoglobulin A measurements. A similar pattern is observed for immunoglobulin G, with significantly lower concentrations in nasal lavage fluid collected using the novel method compared to the conventional method. In contrast, serum and bronchoalveolar lavage fluid samples again show no meaningful differences between the two methods, suggesting that the novel approach consistently reduces contamination in nasal samples and improves the reliability of immunoglobulin G measurements.