Cell tak cell and tissue adhesive

Seahorse XF96 Analyzer (Seahorse Bioscience, Billerica, MA, USA) was used to analyze the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). 96-well Seahorse tissue culturing plates were pre-incubated with Cell-Tak Cell and Tissue Adhesive (354240; Corning, Corning, NY, USA). Cells were seeded in unbuffered DMEM media at a density of 80 K per well. Cells were incubated for 1 hour without CO₂ at 37 °C. OCR and ECAR rates were measured as previous described by us^{79 (link),80 (link)}. The basal OCR and ECAR rates were measured 3 times without adding any inhibitors.

Seahorse XFp cell culture miniplates (Agilent, #103025-100) were treated with Cell-Tak cell and tissue adhesive (0.024 mg/mL Corning, #354240) according to manufacturer specifications and 30,000 cells/well were plated. Agilent Seahorse XF base medium (#103193-100) was supplemented with 25 mM glucose, 2 mM sodium pyruvate, and 2 mM l-glutamine. Basal respiration was normalized by cell count.

A Seahorse Bioscience XFe24 cell culture microplate (Seahorse Bioscience, North Billerica, USA) was coated with Cell-Tak cell and tissue adhesive (Corning, USA) before 50 µl of 22.4 µg ml⁻¹ Cell-Tak solution was added into the well. After 20 min of room temperature incubation, the microplate was washed twice with sterile water. Freshly isolated kidney tubules were resuspended in 37 °C preheated Seahorse XF Assay Medium (Seahorse Bioscience) and seeded onto the coated Seahorse Bioscience XFe24 cell culture microplate at a density of 500 tubules/50 µL/well. A control well had 50 µl Seahorse XF Assay Medium only. Before incubating at 37 °C without CO₂ for 25–30 min, the microplate was centrifuged at 200×g for 1 min. A 130-µl preheated analysis buffer was added to the well after ensuring that all tubules sank to the plate’s bottom; the incubation was continued at 37 °C without CO₂ for 15–20 min. The microplate was then put into a Seahorse XFe24 energy analyzer. An XF Cell Mito Stress Test kit was used to test for mitochondrial stress, an XF Glycolysis Stress Test kit was used to test for glycolysis stress and an XF Palmitate-BSA FAO Substrate was used to test the metabolism of fatty acid oxidation.

Glucose, lactate and glutamate concentrations were measured using enzymatic assays (CMA Microdialysis AB) and a CMA 600 analyzer (Aurora Borealis). Total glutathione was measured using a dedicated quantification kit (Enzo). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed using the Seahorse XF96 plate reader. Briefly, after 24–48 hours of pre-incubation in the indicated medium, 1.5 × 10⁵ HL-60 cells were seeded in 96-well plates coated with Cell-Tak™ Cell and Tissue Adhesive (Corning). After equilibration in unbuffered DMEM with either Gln or Glc (or both) at 37°C in a CO₂-free incubator, OCR and ECAR were measured.

A Seahorse XFe96 culture plate (Agilent Technologies) was coated with 22.4 µg/ml Cell-Tak™ Cell and Tissue Adhesive (Corning®) for one hour at room temperature after which the Cell-Tak was removed and the wells were washed once with 200 µl water. The plate was allowed to air dry. (Cell-Tak was diluted in water, 25 µl of dilution per well, Stock: 1.39 mg/ml). Sorted cMoP were seeded in duplicate with M-CSF (50 ng/ml) and fixed BCG (10⁶/ml) or with M-CSF (50 ng/ml) alone as control. After incubation for 6 days, cells were analyzed by glycolysis stress test on a Seahorse XFe96 Analyzer (Agilent Technologies). Accordingly, Seahorse base media supplemented with 2 mM L-Glutamine, adjusted to pH 7.4 was used as assay medium. Injection plates were prepared with 10 mM Glucose (Port A), 1 µM Oligomycin (Port B) and 50 mM 2-DG (Port C). 2-DG, L-Glutamine Solution and Oligomycin were purchased from Sigma Aldrich. The assay protocols were designed using Wave desktop software (Version: 2.4.0.60 Agilent).