*2.10. Two-Photon Fluorescence Lifetime Imaging Microscopy (FLIM)*

DC were cultured in the presence of HPP (1000 μM) for 6 h prior to stimulation with LPS (100 ng/mL) for 12 h. Two-photon excited NAD(P)H- Fluorescence Lifetime Imaging Microscopy (FLIM) was used to measure the levels of free and protein-bound NADH within these cells, and was performed on a custom multiphoton system (further details regarding experimental setup can be found at the following [15,16]). At least three images for each model were acquired. Afterwards, regions of interest (ROI) were selected, and the NAD(P)H fluorescence decay was analysed.

For the NAD(P)H fluorescence decay analysis, an overall decay curve was generated by the contribution of all pixels in the ROI area. Afterwards, it was fitted with a double exponential decay curve (Equation (1)):

$$I(t) = a\_1 e^{-\frac{t}{\tau\_1}} + a\_2 e^{-\frac{t}{\tau\_2}} + c \tag{1}$$

*I*(*t*) represents the fluorescence intensity at time (*t*) after laser excitation. *α*<sup>1</sup> and *α*<sup>2</sup> represent the fraction of the overall signal comprised of a short and long lifetime component, respectively. *τ<sup>1</sup>* and *τ<sup>2</sup>* are the long and short lifetime components, respectively. *C* corresponds to background light. X<sup>2</sup> is calculated to evaluate the goodness of multiexponential fit to the raw fluorescence decay data—the lowest χ<sup>2</sup> values were considered in this study.

For NAD(P)H, a two-component fit was used to differentiate between the free (*τ*1) and protein-bound (*τ*2) NAD(P)H. The average lifetime (*τavg*) of NAD(P)H for each pixel is calculated by a weighted average of both the free and bound lifetime contributions (Equation (2)):

$$\pi\_{avg} = \frac{(\alpha\_1 \times \pi\_1) + (\alpha\_2 \times \pi\_2)}{(\alpha\_1 + \alpha\_2)} \tag{2}$$

#### *2.11. Metabolic Profiling Using Seahorse Analysis*

DC were cultured in the presence of IP or HPP (both 1000 μM) for 6 h prior to stimulation with LPS (100 ng/mL) for 12 h. The cell culture medium was replaced with complete XF assay medium (pH of 7.4, supplemented with 10 mM glucose, 1 mM sodium pyruvate, 2 mM L-glutamine) and DC were then transferred at a density of 2 × <sup>10</sup><sup>5</sup>

cells/well to a Seahorse 96-well microplate, which was coated with Corning™ Cell-Tak Cell and Tissue Adhesive and incubated in a non-CO2 incubator. Blank wells were prepared containing XF assay medium only to subtract the background oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) during analysis. Oligomycin (1 mM), FCCP (1 mM), rotenone (500 nM), antimycin A (500 nM), and 2-DG (25 mM) were prepared in XF assay medium. Inhibitors were loaded into the appropriate injection ports on the cartridge plate and incubated for 10 min in a non-CO2 incubator at 37 ◦C. Oligomycin, FCCP, rotenone and antimycin A, and 2-DG were sequentially injected while the OCR and ECAR readings were simultaneously measured. Wave software (Agilent Technologies, Santa Clara, CA, USA) was used to analyse the results. The rates of basal glycolysis, max glycolysis, glycolytic reserve, basal respiration, max respiration, and respiratory reserve were calculated as detailed in the manufacturer's protocol and as supplied in Supplementary Table S1.
