Lactate assay kit

Lactate dehydrogenase (LDH) and lactate in cell medium were measured with Cytotoxicity Detection Kit (LDH) (Roche) and Lactate Assay Kit (Merck), respectively. The total protein content was determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). All assays were performed according to the manufacturer’s instructions. The DNA content was determined with a DNA assay based on the fluorescent DNA dye Hoechst 33342 (Thermo Fisher Scientific) as described (Rajh et al. 2016 (link)). Briefly, after lysis with 0.04% (wt/vol) SDS in water cell lysates were transferred to a 96-well microplate, diluted to 100 μL with water and mixed with 100 μL of Tris-NaCl buffer (50 mM Tris, 100 mM NaCl, pH 8.3) with 10 μg/mL Hoechst 33342. Samples were incubated for 15 min at room temperature and then Hoechst fluorescence was measured with VICTOR microplate reader (PerkinElmer) using 355 nm excitation filter and 460 nm emission filter.

Levels of IL-6, chemokine (C-X-C motif) ligand-1 (KC), TNF-α, IL-1β, IL-10 and leptin in cell culture supernatant fractions were measured with murine DuoSet ELISA Development kits (R&D Systems, Abingdon, UK). An enzymatic assay adapted from the Lactate Assay kit (Merck) was used to determine lactate levels.

Lactate and pyruvate concentrations were determined using Lactate Assay Kit and Pyruvate Assay Kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Cells grown in 250 mL Greiner flasks were incubated with 50 µM SIL or ctrl for 24 h, when they had reached a confluency of 70–80%. After 24 h cells were harvested from the flasks, washed once with PBS and stored at −80 °C. On the day of experimentation, samples were thawed and heated to 95 °C for 10 min, before the Assay kit was performed. Absorbance was measured using a ClarioStar plate reader (BMG Labtech, Ortenberg, Germany). Samples were measured in duplicates.

Cells were seeded in culture dishes and cultured for 8 h. The culture medium was then changed and cells were incubated for an additional 16 h. Subsequently, the culture medium was collected for determination of glucose concentration and lactate levels using a Glucose assay kit (Amplite ™ Glucose Quantitation Assay kit, AAT Bioquest, Sunnyvale, USA) and a Lactate assay kit (MAK064, Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Glucose consumption was calculated as the difference in glucose concentration between fresh medium and cell supernatant. Lactate production was determined as the difference in lactate concentration between cell supernatant and fresh medium. Data were normalized to final cell counts and incubation time.

The workflow for accumulation experiments is shown in Figure 2. Cells were seeded into 6-well plates two days prior, reaching 80–90% confluency on the day of the experiment. For the experiment, growth medium was withdrawn and cells were washed once with the respective medium, before fully supplemented MEM or glucose-reduced DMEM were added, containing 1MBq/mL 2-[¹⁸F]FDG (n = 5). Then, 1 h after addition of 2-[¹⁸F]FDG, aliquots of the supernatants were taken, cells were washed two times with PBS, detached with 500 µL of Accutase and resuspended in MEM. A 100 µL aliquot of the cell suspension was measured with a gamma counter (PerkinElmer, 2480 Automatic Gamma counter, Wizard23) and subsequently, cells were mixed with trypan blue and counted with a Neubauer chamber. Values are expressed as % applied dose (% AD) per 10⁴ cells.
The aliquots of the supernatants were filtered through a Microcon-30 kDa Centrifugal Filter Unit (Merck KGaA, Darmstadt, Germany) to remove LDH from the medium. Then, lactate concentration was determined with a lactate assay kit (Merck KGaA, Darmstadt, Germany) following the supplier’s instructions.

One hour post-gentamicin treatment, hPMN were lysed in triton X-100 (0.2%) for 10 min and assayed using the Lactate Assay Kit (Merck) as per the manufacturer’s guidelines.