Sodium dl lactate solution

All cultures were grown at 30°C and shaken at 250 rpm. Nitrate reducing minimal media was prepared with the following final concentrations: NaCl (0.06 mM), NH₄Cl (1.4 mM) (for ammonium replete conditions but not used in NH₄⁺-free conditions), MgCl₂ (0.2 mM), CaCl₂ (0.04 mM), KCl (0.1 mM), K₂HPO₄ (1.1 mM), NaHCO₃⁻ (30 mM), cysteine (1 mM) as reducing agent, resazurin as redox indicator, and trace elements and trace vitamin solutions as reported (Yoon et al., 2013 (link); Hillesland et al., 2014 (link)). 1 M sterile filtered (0.2 μm) concentrated stocks of 60% w/w sodium DL-lactate solution (Sigma-Aldrich, St. Louis, MO, United States), sodium-nitrate and sodium-nitrite (≥ 99%, Fisher Scientific, Pittsburg, PA, United States) were diluted into media prior to autoclaving to achieve the desired C:NO₃⁻ ratio. C:NO₃⁻ ratio was calculated based on (Yoon et al., 2015a (link)) where the number of C atoms (n) in the e-donor is multiplied by the concentration of the e-donor, divided by the number of N atoms in the e-acceptor multiplied by the concentration of the e-acceptor (Supplementary Table S1). See SI Materials and Methods for complete description of Hungate technique prepared media. Mean pH for all culture vessels (time series and end-point; Supplementary Table S2), measured at the end of each experiment, was 7.3 ± 0.05 (n = 144).

Cells were plated onto PLB-coated Lab-Tek II imaging chambers (Nunc), and all experiments were performed at 37 °C, 5% CO₂ in tissue culture incubators. Cells were fixed with the addition of paraformaldehyde to a final concentration of 4% and incubated at 37 °C for 15 mins. Cells were then washed in DPBS with the serial addition and removal of 500 μL DPBS (5 time; 200 μL of liquid was always maintained within the well to prevent desiccation of the PLB). Cells were permeabilised, blocked and labelled as above. For live cell imaging, cells were resuspended in growth media, and added to wells containing PLBs that contained an equal volume of DPBS. Imaging proceeded at 37 °C, 5% CO₂. For single-colour STORM imaging, slides were immersed in 0.22 μm-filtered STORM imaging buffer (560 μg/mL glucose oxidase, 34 μg/mL catalase, 1% β-mercaptoethanol, 25 mM glucose, 5% glycerol (all from Sigma-Aldrich), and 25 mM HEPES/DPBS pH 8 (Thermo Fisher Scientific), refreshed regularly to maintain a low-oxygen environment for optimal fluorophore blinking. For two-colour STORM imaging, slides were immersed in 0.22 μm-filtered OxEA imaging buffer (50 mM β-MercaptoEthylamine hydrochloride (MEA, Sigma-Aldrich), 3% (v/v) OxyFluor™ (Oxyrase Inc.), 20% (v/v) sodium DL-lactate solution (Sigma-Aldrich) in DPBS, pH adjusted to 8–8.5 with NaOH), as previously published^{64 (link)}.

From day 16 to 18, the cultures were fed with lactate medium composed of DMEM (no glucose) supplemented with 1% sodium DL-lactate solution (60%, Sigma Aldrich) and 25 μg/mL of gentamicin [22 (link)]. For the first 2 days of purification, the medium was replenished daily. Thereafter, medium exchanges occurred every other day. Lactate medium-based purification was performed for a maximum of 7 days. Cardiac clusters were then dissociated with 0.5% trypsin (Sigma Aldrich) and processed for flow cytometry analysis to determine the amount of cardiac troponin T (cTnT) positive cells as a marker for cardiomyocyte content. Subsequently, the cells were plated in DMEM with 10% FBS and 25 μg/mL of gentamicin onto 0.1% gelatin-coated plates. After 2 days in culture, cultures containing ≥ 90% cTnT positive cells on the day of dissociation were maintained in DMEM (low glucose) supplemented with 2.5% FBS and 25 μg/mL of gentamicin until further analysis. Cultures yielding ≤ 90% cTnT positive cells after 7 days of treatment with lactate medium were subjected to additional lactate medium-based purification for 3–5 days before they were also switched to DMEM (low glucose) supplemented with 2.5% FBS and 25 μg/ml gentamicin.

As the analyzed population showed only an insufficient purity of 78% iPS-CMs metabolic purification applying lactate was performed as previously described (26 (link)). Briefly, cells were reseeded in lower density in RPMI supplemented with insulin, 20% Fetal Bovine Serum (FBS) and 5 μM Pro-survival Compound, DDD00033325 (Calbiochem) by applying 0.25% Trypsin/EDTA. Cells were allowed to recover for 4 days and were then cultivated in RPMI without Glucose and HEPES supplemented with 4 mM Sodium DL-lactate solution (Sigma) for 4 days. Afterwards, media was changed back to RPMI with insulin. All subsequent analyses were performed at a cell density of 37 500 cells/cm².

Spermatozoa were purified using a 60% Percoll^® (Sigma-Aldrich, St Louis, MO, United States) gradient equilibrated with Hepes-buffered Ham’s F10 medium (Gibco, Life Technologies, United Kingdom) supplemented with sodium bicarbonate (0.2% NaHCO3; w/v; Merck, Darmstadt, Germany), sodium pyruvate (0.003% C₃H₃NaO₃; w/v; Sigma-Aldrich) and sodium DL lactate solution (0.36% v/v, Sigma-Aldrich). In order to ensure the complete elimination of the gradient material, the recovered sperm cells were washed twice with supplemented Hepes medium and centrifuged at 400 g for 10 min. This procedure allowed the purification of the samples from cells other than sperm while maintaining proportions of sperm subpopulations similar to the native semen sample (Mengual et al., 2003 (link); Zhao et al., 2007 (link); Wang G. et al., 2013 (link)). The sperm purification efficiency was checked using phase-contrast microscopy. All purified sperm samples contained <1% of potential contaminating cells. For each sample, an aliquot of 20 million of DGC sperm was taken for subsequent procedures of protein solubilization, while the remaining material was subjected to incubation with capacitation medium.

Semen samples were obtained from 30 healthy adult Holstein Friesian bulls at a local breeding facility (Slovenské biologické služby, a.s., Nitra, Slovakia) with the help of an artificial vagina. All samples (1 sample per bull) were distributed into three fractions. The first and second ones were immediately transported into the laboratory and incubated for 30 min at 39 °C and 5% concentration of CO₂, either with physiological saline solution (IMUNA PHARM, A. S., Šarišské Michaľany, Slovakia) as a control or with a capacitation medium consisting of 100 mM sodium chloride (Sigma-Aldrich, St. Louis, MO, USA), 3 mM potassium chloride (Sigma-Aldrich, St. Louis, MO, USA), 25 mM sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 283 µM sodium phosphate (Sigma-Aldrich, St. Louis, MO, USA), 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 1.5 mM magnesium chloride (Sigma-Aldrich, St. Louis, MO, USA), 2.5 mM calcium chloride (Sigma-Aldrich, St. Louis, MO, USA), 0.37% sodium DL-lactate solution (60%; Sigma-Aldrich, St. Louis, MO, USA) and 0.2% phenol red solution (0.5%; Sigma-Aldrich, St. Louis, MO, USA) [84 (link)] diluted in a ratio of 1:40. The third part was cryopreserved for further investigation.