README - Chapter 5 (Results Chapter 3) Documentation 

Project Title 
Investigating the role of serotonergic receptor subtypes in the modulation of cell-specific immune function

Dataset DOI: https://doi.org/10.5258/SOTON/PG/D228

Author
Jamie Thomas

Institution 
University of Southampton 

Supervisor 
Professor Diego Gomez-Nicola
Dr Max Ahmed 
Dr Gary Gilmore

Date
April 2026

Overview
This folder contains the dataset(s), supporting materials, and analysis files used in Chapter 5 of the PhD thesis. The purpose of this README is to document the contents, structure, and usage of the data for transparency, reproducibility, and future reference.

Purpose
This data was used to investigate the impact that serotonergic signalling had on immune cells within an in vivo environment, in addition to how this can affect behavioural outcomes associated with sickness behaviour. The techniques utilised included flow cytometry, ELISA, and behavioural assessments (i.e., weighing, burrowing, sucrose preference test, grooming, head twitch). 

Structure and Details

1. Model Optimisation 
Data Folders (5) - Antibody Incubation, Antibody Panel Test, LPS Administration, Sorting Experiment, and Training. 'Antibody Incubation' includes experiments in which flow staining was conducted for 18h and 30m to compare staining effectiveness, while 'Antibody Panel Test' relates to the testing on antibodies within the actual antibody panel. The 'Sorting Experiment' is an extension of this, again highlighting the antibody panel being used. The 'Training' is associated with the training procedures and preliminary results associated with the model, while the 'LPS administration' folder contains the behavioural and flow cytometry data associated wth the initial dosage of LPS to the animals. 
Data obtained from behavioural assessment (i.e. weight change, burrowing) and flow cytometry experiments
Key variable = assess sickness behaviour and immune population within the in vivo model 
Flow cytometry data were analysed in FlowJo and extracted to Excel and PRISM for analysis. Behavioural data was manually recorded and then analysed in Excel/PRISM. 
Graphs used to visualise the data 
Note: limited anatomical regions for immune population analysis, restrictive stimulation 

2. LPS and Psilocybin 
Data Folders (3) - Experiment 1-3, and Data Files (5)
Data folders contain the flow cytometry data for experiments 1-3, while the data files include an excel summary table draft and final version and a PRISM files with data analysis included. 
Data obtained from flow cytometry experiments
Key variable = assess viability and immune populations within the in vivo culture following stimulation and serotonergic receptor agonist treatment + assessment of behaviour. 
Flow cytometry data was analysed in FlowJo and extracted to Excel and PRISM for analysis. Behavioural assessments were manually recorded.
Graphs used to visualise the data
Note: specific stimulation utilised, limited immune cells within the scope, variable levels of immune cells, limited behavioural assessments utilised

2.1. Psilocybin Administration 
Data Files (2) - Excel and PRISM
Data files contain the summary of the head twitch and grooming behaviour outputs collected by Monica, while the prism file is the associated analysis of this.  
Data obtained from manual collection and observations
Key variable = assess head twitch response and grooming following treatment with psilocybin and/or LPS.
Behavioural assessments were visually recorded and manually counted.
Graphs used to visualise the data
Note: specific stimulation utilised, limited behavioural assessments utilised

3. LPS and 5-HTP
Data Folders (2) - Experiment 1-2, and Data Files (7)
Data folders contain the flow cytometry data for experiments 1-2, while the data files include an excel summary table for the behaviour and the immune cell analysis and a PRISM files with data analysis included for each of these. 
Data obtained from flow cytometry experiments and behaviour assessment from manual collection
Key variable = assess viability and immune populations within the in vivo culture following stimulation and serotonergic receptor agonist treatment + assessment of behaviour. 
Flow cytometry data was analysed in FlowJo and extracted to Excel and PRISM for analysis. Behavioural assessments were manually recorded.
Graphs used to visualise the data
Note: specific stimulation utilised, limited immune cells within the scope, variable levels of immune cells, limited behavioural assessments utilised

3.1. 5-HTP Administration
Data Files (5) - Excel and PRISM
Data files contain the raw data of the 5-HT ELISA, alongside two files of the analysed data. Also included are the PRISM files of the post-analysed data. 
Data obtained from ELISA
Key variable = assess serotonin levels in the brain, spleen, serum following treatment with a range of 5-HTP concentrations
ELISA data was recorded using a plate reader and analysed in Excel based on the producer's instructions 
Graphs used to visualise the data
Note: limited range of 5-HTP, selective time point assessed, no comparison within LPS model.

4. Cytokine PCR
Data Files (8) - Excel and PRISM
Data files relate to the PCR data for each of the genes of interest and the housekeeper gene, alongside a data analysis PRISM file. 
Data obtained from RT-PCR experiments.
Key variable = assess gene expression in spleen tissues following treatment with LPS and/or serotonergic agonists.  
RT-PCR data was collected by PCR machine and then CT values were analysed in excel using delta-deltaCT.
Graphs used to visualise the data
Note: limited genes for analyse, suboptimal timepoint for gene analysis

5. Cytokine Mesoscale
Data Files (2) - Excel and PRISM
Data files contain mesoscale data collected by Monica, while the prism file is the associated analysis of this.  
Data obtained from mesoscale multiplex analysis 
Key variable = assess cytokine levels within the serum following LPS and/or serotonergic agonist treatment 
Mesoscale data was obtained from a plate reader and analysed in excel. 
Graphs used to visualise the data
Note: limited cytokines observed, limited sample location assessments 

Contact
Name: Jamie Thomas
Email: jlt1n21@soton.ac.uk 