A total of 4 × 5 ml EDTA blood tubes were collected from each of five donors. Tubes were processed immediately or refrigerated for 6, 12 or 24 h before processing. All PBMCs were cryopreserved and subsequently thawed and assessed by fluorescence flow cytometry for FSC and SSC to distinguish monocytes and lymphocytes, and expression of CD3, CD4 and CD14. (A) Viability was determined using NIR viability dye, with representative dot plots showing changes in forward scatter vs viability over time. Within viable cells, (B) monocytes were gated as CD4^loCD14⁺ and their identity confirmed by FSC/SSC, (C) B and NK cells were gated collectively as CD3-negative lymphocytes, (D) CD4⁺ T cells were gated as CD3⁺CD4⁺ and (E) CD8⁺ T cells were identified in this limited panel as CD3⁺CD4⁻. Data were analysed with a one-way repeated measures ANOVA with post-hoc Tukey’s tests and P values indicating significant differences between the timepoints are shown.