Nile red

After 24 h treatment with or without 100 µM NMN in HN and HH conditions, HepG2 cells and C2C12 myotubes were harvested and washed twice in cold PBS and incubated with different fluorescent dye solutions in culture media without phenol red and serum for 30 min at 37 °C in the dark. The fluorescent markers used were as follows: 10 nM NAO (sc-214487, Santa Cruz Biotechnology, Dallas, Texas, USA) for mitochondrial mass, 25 nM MitoView Red for mitochondrial function, 2 µg/mL JC-1 for mitochondrial membrane potential, 2.8 µg/mL DHR123 for mitochondrial ROS, and 100 ng/mL Nile Red (7726.1, Carl Roth, Karlsruhe, Germany) for neutral and polar lipids. After dye incubations, cells were washed with PBS and resuspended in 500 μL culture media without phenol red. The stained cells were analyzed on a Cytomics FC 500 flow cytometer (Beckman) (FL1/FL2/FL3/FL4/FL5) using Flowing software version 2.5.1. For each sample, three replicates of 15,000 events each were acquired.

The lipid staining of bacteria was performed similar to previous protocols [17 (link),19 (link),26 (link)]. A mid-logarithmic growth-phase culture was diluted in PBS to 2.5 × 10⁸ CFU/mL. 200 µL of these cell-suspension were incubated with peptide concentrations 2–3 times lower than their LC_99.9% for 20 min at 37 °C and 300 rpm, before being stained with 10 µg/mL Nile Red (Sigma Aldrich, Steinheim, Germany), a fluorescent membrane dye. Nile Red stock solution of 1 mg/mL was prepared in methanol (Carl Roth, Karlsruhe, Germany). After centrifugation, 1 µL of the cell pellet was mounted onto a microscope slide with agar (2 w%) and covered with a coverslip of 0.17 µm thickness (Menzel, Inc., Spartanburg, SC, USA).
Microscopy was performed using a Leica SP5 confocal microscope (Leica Microsystems, Inc., Singapore) with spectral detection and a Leica HCX PL APO CS 63x NA 1.4 oil immersion objective. Nile Red was excited at 561 nm and fluorescence emission was detected between 570–750 nm. A 488 nm laser line was additionally activated for simultaneous acquisition of fluorescence and transmission data. Images were recorded using 47 × 47 nm sampling (x/y).

One hundred microliters of EVs was mixed with 4 μL of Nile Red (2.5 mg/mL in acetone, Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Simultaneously, a control probe was prepared by mixing 100 μL of PBS with 4 μL of Nile Red. After 15 min of incubation in the dark and at RT, the samples were transferred into centrifugal filters with a 10 kDa cut-off. Next, the samples were washed 6 times with 400 μL of PBS to rinse off the redundant dye. The control probe was needed to monitor whether the entire free Nile Red was washed out from the samples. A small droplet (10 μL) of the finally obtained stained sample was placed on the microscope slide glass and covered with a coverslip. Imaging was taken under a fluorescence microscope (Nikon Eclipse Ni with color camera combined with the software NIS Elements Basic Research version 5.30, Precoptic, Warsaw, Poland) with a red filter block (FF01-474/27 nm Excitation, FF02-525/45 nm Emission), as Nile Red is excited by green light and emits red light, under the magnification of 1000× with oil immersion (Nikon, Plan Apo VC objective lens 100×/1.40 Oil OFN25 DIC N2).

Nile Red (#7726, Roth) was dissolved to 0.5 mg/mL in acetone. The solution was vortexed and left shaking overnight, in the dark. A 1 μg/mL working stock was prepared in 1x PBS and stored at 4 °C in the dark. Prior to starting the experiment, Nile Red was added to S-complete or molten NGM to a final concentration of 0.02 μg/L. Worms at the L1 stage were added to the S-complete (in 10 μL M9 buffer) or directly to the surface of seeded NGM which had been supplemented with Nile Red and the compound of choice. The plates were incubated at 20 °C for 48 h and then analysed using the CLARIOstar®.
The Nile Red fluorescence was read with an excitation wavelength of 528nm and an emission wavelength of 580nm, with GFP fluorescence (a measure of the number of worms based on the myo-3::gfp reporter) measured at an excitation and emission wavelength of 482nm and 515nm, respectively. For both spectrums, an 8nm slit was used to ensure a narrow wavelength of light was used to excite the fluorophores. To compare the amount of Nile Red in each well a normalisation calculation was applied, where the presence of the GFP reporter in the body wall muscle from the strain AW306 was used to normalise to the number of worms. The Nile Red reading was then divided by the normalised GFP value to obtain a normalised Nile Red value.