Seahorse xf base medium without phenol red

For Mito Stress testing, αA5^KD cells and their scrambled controls were plated 6 h before the assay onto Seahorse cell culture microplates (Agilent Technologies, Santa Clara, CA; Cat#101085-004). The four corner wells of the plate were used as negative controls. The assay medium consisted of Seahorse XF base medium without phenol red (Agilent Technologies, Cat#103335-100), supplemented with glucose (25 mM), sodium pyruvate (1 mM), and glutamine (2 mM). Oligomycin (1 µM), FCCP (2 µM), and rotenone/antimycin (0.9 µM) were used as stressors (Agilent Technologies, Cat#103015-100). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at basal level and after addition of different stressors using a Seahorse XFe96 analyzer (Agilent Technologies). Assay operation software Wave version 2.6.1 was used to run the assay and collect the data. For normalization, live cells stained with Hoechst 33342 (1:2,000 dilution) (ThermoScientific, Cat#62249) were counted using a Cytation 5 plate reader (BioTek Instruments - Agilent). Values obtained from the Mito Stress test were normalized per 1,000 cells.

Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using an XF96 extracellular flux analyzer (Seahorse Bioscience) as previously described^{124 (link),127 (link)}. XF96 cell culture plates were coated with Cell-Tak (Corning, Cat. # 354240) to enhance immobilization of samples at the bottom. Typically, one eye organoid contains 10 thousand cells. The organoids were washed and resuspended with Seahorse XF base medium without phenol red (Agilent, Cat# 103335-100) supplemented with L-Glutamine (2 mM; Millipore Sigma, Cat. #G8540). Five to eight organoids per well were plated onto the XF96 plates and spun down at 400 × g for 2 min without deceleration. Prior to performing the assay, plates were incubated at 37’C without CO₂. First, 10 mM glucose or lactate were injected to the seahorse microplates. After three initial readings, oligomycin (2 μM; Agilent, Cat. #103015-100) was injected into each well. Next, to monitor glycolytic activity, 2-DG (25 mM) or GNE-140 (20 µM) were added to the plate followed by three readings to complete ECAR and OCR tracing.

Cells were seeded at 1 × 10⁴ per well of the Seahorse XFp Cell Culture Miniplate (Agilent, Santa Clara, CA, USA) one day prior to measurement. Cells were incubated with the Seahorse XF Base Medium (without phenol red) (Agilent) supplemented with 10 mM glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, and 5 mM HEPES-NaOH pH 7.4 for 1 h at 37 °C in a CO₂-free incubator. The oxygen consumption rate (OCR) of the cells was measured by the Seahorse XFp Extracellular Flux Analyzer (Agilent) according to the manufacturer’s protocols. Oligomycin (1 µM), FCCP (0.5 µM for A549 cells or 1.0 µM for the other six cell lines), and rotenone/antimycin A (0.5 µM each) were serially injected into the well, and the OCR was measured for one min three times at each step. The OCR was analyzed by Seahorse Wave software (Agilent).