Te2000 inverted fluorescence microscope

To determine the percentage of spermatozoa with EGFP in their acrosomes, 10 μL aliquots of the sperm suspensions were placed on poly-l-lysine-coated glass slides and covered with coverslips. To detect the presence of EGFP in the acrosomes, the cells were quantified using a Nikon TE2000 inverted fluorescence microscope with fluorescence optics (excitation 480 nm, emission 515 nm) as previously described (Buffone et al., 2009a (link), b (link)). In other experiments, acrosome reaction was quantified as previously described using flow cytometry using a FACSCanto II flow cytometer (BD) [21]. The viable sperm were selected by staining with propidium iodide (final concentration, 10 μg/ml), and their acrosomal integrity was determined by the presence of acrosomal EGFP as previously reported (Muro et al., 2012 (link)).

Characterization of MSCs from murine bone marrow was assessed by flow cytometry [4 (link)] using a FACScalibur instrument (Becton Dickinson, San Diego, CA). Cells were washed in Hank’s balanced salt solution containing 0.1% bovine serum albumin and 0.1% sodium azide (NaN₃) and labeled with appropriately diluted antibodies (CD44, Sca-1, isotype controls) directly conjugated with fluorescein, for 30 minutes, followed by three washes and fixation in 2% paraformaldehyde. Data analysis used Cell Quest Software (Becton Dickinson). Phalloidin was used for labeling filamentous actin. Cultured cells were washed in phosphate buffered saline (PBS) and fixed in 3.7% formalin for 10 min. After three washes with PBS, the cells were permeabilized with 0.5% Triton X-100 (Sigma) for 20 min. These cells were stained in the dark with TRITC-conjugated phalloidin antibody (Molecular Probes, Eugene, OR) for 30 min, washed and mounted on microscope slides with Fluoroshield mounting medium (Abcam, Cambridge, UK). Images were acquired on a Nikon TE2000 inverted fluorescence microscope using a Spot 12-bit 1600 × 1200-pixel charge-coupled device; red fluorescence used excitation 536–556 nm, a 580 nm dichroic filter. Images were processed with ImageJ software (National Health Institute, Bethesda, USA)