Ethd 1

The cell survival rate in the 3D cell constructs was assessed at one and three days after fabrication. A fluorescent live/dead staining solution (Thermo Fisher) was used. Each 3D cell construct and the 2D cultured cells were washed in Dulbecco’s PBS (DPBS) three times before staining. The DPBS mixture with Calcein-AM (2 µM) and EthD-1 (4 µM) was filtered through a 0.22-µm syringe filter (Sigma-Aldrich). Cell morphologies were observed under a fluorescence microscope (Leica DMi8, Leica). Three independent samples were analyzed.

Plasma membrane integrity and acrosome integrity were evaluated by staining the spermatozoa with Ethidium homodimer-1 (EthD-1, Molecular probes Inc., Eugene, OR, USA) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA, Sigma, St. Louis, MO, USA) (Kitiyanant et al., 2002 (link)). In brief, an aliquot of 5 μL sperm suspension was mixed with an equal volume of 2 μM EthD-1 for 10 min at 37°C. Subsequently, a 5 μL of a 10 mg/mL salmon sperm DNA (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in phosphate buffered saline (PBS) was added to bind excessive EthD-1 for 3 min at 37°C. After centrifugation, the spermatozoa were then smeared on a glass microscopic slide and fixed in 95% (v/v) ethanol for 30 s. Fixed spermatozoa were stained with 100 μg/mL FITC-PNA in PBS in a humidified chamber for 30 min at 4°C then rinsed with 4°C distilled water and allowed to air dry at 4°C. The fluorescent labeled slides were kept in the dark until evaluation. A total of 200 spermatozoa per slide were randomly visualized using an epifluorescent microscope at 1,000× and then classified into 3 categories as previously described (Cheng et al., 1996 (link)).