Sadie rose Grant The decision to wash the cecum tissue with phosphate-buffered saline (PBS) versus taking the cecum contents straight from the GI tract can depend on the specific goals of your experiment and the nature of the microbial community you are trying to study.

Here are some considerations:

1. Sample composition: Washing with PBS can help remove transient bacteria and mucus, thereby allowing you to focus more on the microbes tightly adhering to or embedded within the gut lining. This could be beneficial if you're interested in the interaction between the host and microbiota. However, if you want a more comprehensive view of the total microbial community, including transient and luminal bacteria, you might prefer to avoid washing and take the contents directly.

2. Downstream analysis: Consider how washing might impact your downstream analysis. For instance, washing with PBS could potentially dilute the proteins you are trying to analyze, making it more challenging to detect low-abundance species. However, it might also reduce potential contaminants, making your proteomics data cleaner.

3. Sample handling consistency: It's crucial to handle all samples in a consistent manner to avoid introducing unnecessary variability into your dataset. If you're comparing cecum contents with other types of samples that are washed with PBS (like fecal samples), it might make sense to wash the cecum as well.

4. Study Reproducibility: If you're building upon previous work, consider the methods used in those studies. Replicating their methods can help make your results more comparable.

As for the ideal way to handle mouse cecum samples in bottom-up proteomics, it will ultimately depend on the specific scientific question you want to answer, and it may be worth testing both methods (i.e., with and without washing) on a small number of samples to see how much of an effect it has on your results. Also, make sure to control for any potential effects by including appropriate reference samples and replicate measurements.

It's also important to remember that the state of the cecum (e.g., whether it's relaxed or contracted) can also affect the amount of luminal content and, thus the microbial composition, so careful and consistent sample collection is crucial.