README - Chapter 4 (Results Chapter 2) 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 4 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 vitro environment. The techniques utilised included flow cytometry, ELISA, and multiplex analysis. 

Structure and Details

1. General Culture Optimisation 
Data Folders (3) - Cell Density and Media Optimisation, Data Analysis, and Stimulation Optimisation 
'Cell Density and Media Optimisation' includes the core number and media experiments included within the thesis - containing the flow cytometry data for all + analysis. 'Stimulation Optimisation' includes the flow cytometry data for 24h and 28h stimulations, alongside the use of other stimulation techniques. 'Data Analysis' includes the summary data table and the statistics associated with the optimisations included within the thesis. 
Data obtained from flow cytometry experiments
Key variable = assess viability and immune populations within the in vitro culture at different timepoints and with different cell densities, media composition, and stimulation. 
Flow cytometry data was analysed in FlowJo and extracted to Excel and PRISM for analysis. 
Graphs and pie charts used to visualise the data 
Note: specific stimulation utilised, limited immune cells within the scope, variable levels of immune cells

2. Immune Cell Populations
Data Folders (7) - Drug 1-7
Data folders contain the flow cytometry data for experiments 1-3, alongside an excel summary table and a PRISM file with data analysis included. Drugs 1-7 include 8-OH-DPAT, 25CN-NBOH, BW723C86, Lorcaserin, AS-19, Psilocin, and Serotonin.
Data obtained from flow cytometry experiments
Key variable = assess viability and immune populations within the in vitro culture following stimulation and serotonergic receptor agonist treatment. 
Flow cytometry data was analysed in FlowJo and extracted to Excel and PRISM for analysis.
Graphs and pie charts used to visualise the data
Note: specific stimulation utilised, limited immune cells within the scope, variable levels of immune cells

3. Annexin V
Data Folders (3) - Experiment 1/2 
Data folders contain the flow cytometry data for experiments 1-2, alongside an excel summary table and a PRISM file with data analysis included. 
Data obtained from flow cytometry experiments
Key variable = assess viability, necrosis and apoptosis within immune populations within the in vitro culture following stimulation and serotonergic receptor agonist treatment.
Flow cytometry data was analysed in FlowJo and extracted to Excel and PRISM for analysis.
Graphs and pie charts used to visualise the data
Note: specific stimulation utilised, limited immune cells within the scope, variable levels of immune cells, absence of NK cell populations 

3. BigFoot - Subtype and Activation 
Data Folders (6) - Experiment 1-4 and Bigfoot Optimisation
Data folders contain the flow cytometry data for experiments 1-4, and optimisation experiments, alongside an Excel summary table.
Data obtained from flow cytometry experiments
Key variable = assess viability, immune populations, and activation and exhaustion markers within the in vitro culture following stimulation and serotonergic receptor agonist treatment.
Flow cytometry data was analysed in FlowJo and extracted to Excel and PRISM for analysis.
Graphs and pie charts used to visualise the data
Note: specific stimulation utilised, limited immune cells within the scope, variable levels of immune cells, limited activation and exhaustion markers utilised

4. Mesoscale 
Data Files (5) - Excel and PRISM 
Data files include 'Data analysis_MSD_JN' and 'Data analysis_MSD_corrected_JN' are the raw data formats. The 'Mesoscale Data Table' provides the summarised data for analysis, while the Mesoscale 1/2 PRISM documents was the associated analysis. 
Data obtained from multiplex analysis 
Key variable = cytokine levels within the in vitro culture following stimulation and serotonergic receptor agonist treatment.
Mesoscale data was analysed by MSD and provided in Excel format; the values were extracted and analysed in PRISM. 
Graphs used to visualise the data
Note: specific stimulation utilised, general culture assessment not immune cell specific, limited number of cytokines assessed. 

5. Serotonin ELISA
Data Files (3) - Excel and PRISM
Data files include '5-HT ELISA Raw Data' is the raw data formats. The '5-HT Culture Supernatant Analysis' provides the summarised data for analysis, while the '5-HT ELISA Supernatant' PRISM file was the associated stat analysis.
Data obtained from ELISA 
Key variable = serotonin levels within the in vitro culture following stimulation and serotonergic receptor agonist treatment.
ELISA obtained from a plate reader and read and stored within Excel for analysis. Analysis was conducted within PRISM.
Graphs used to visualise the data
Note: specific stimulation utilised, general culture assessment not immune cell specific.

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