Quant it protein assay

The cells were washed with phosphate‐buffered saline (PBS), and the culture medium was replaced with Advanced RPMI 1640 medium (Gibco) for PC3M and PNT2 cells, containing an antibiotic‐antimycotic mix and 2 mM L‐glutamine. EVs from the conditioned medium were isolated by a differential ultracentrifugation protocol, as we previously reported (Yokoi et al., 2017 ). Briefly, the conditioned medium was centrifuged at 2000 × g for 10 min to remove contaminating cells. The resulting supernatants were then transferred to fresh tubes and filtered through a 0.22 μm filter (Millipore, Billerica, MA). The filtered conditioned medium was centrifuged for 70 min at 110,000 × g using a SW41Ti rotor to pellet the enriched EVs (Beckman Coulter, Rea, CA). The pellets were washed with 11 mL of PBS and ultracentrifuged at 110,000 × g for another 70 min using the SW41Ti rotor. The EV pellets were stored in a refrigerator at 4°C until use. The fraction containing the EVs was measured for its protein content using the Quant‐iT Protein Assay with the Qubit2.0 Fluorometer (Invitrogen, Carlsbad, CA) or Micro BCA protein assay kit (Thermo Scientific, MA, USA). The particle count and size distribution of EVs were determined using NanoSight system (NanoSight Ltd, Amesbury, UK). Samples diluted 100‐fold with PBS were analysed using the NanoSight system LM10 with NTA2.3 Analytical software (NanoSight).

The cells were washed with phosphate-buffered saline (PBS), and the culture medium was replaced with advanced Dulbecco’s Modified Eagle Medium for HCT116 cells, WiDr cells, SW1116 cells, HT29 cells and CCD-18Co cells, or advanced RPMI medium for the other cell lines, containing an antibiotic–antimycotic and 2 mM L-glutamine (but not containing FBS). After incubation for 48 h, the CM was collected and centrifuged at 2,000 g for 10 min at 4 °C. To thoroughly remove cellular debris, the supernatant was filtered through a 0.22 μm filter (Millipore). The CM was then used for EV isolation. To prepare EVs, CM or the sera from colorectal patients and healthy donors were ultracentrifuged at 110,000 g for 70 min at 4 °C. The pellets were washed with 11 ml of PBS, ultracentrifuged at 110,000 g for 70 min at 4 °C and resuspended in PBS. The putative EVs fraction was measured for its protein content using a Quant-iT Protein Assay with Qubit2.0 Fluorometer (Invitrogen).

For the analysis of tyrosine phosphorylation during the late stages of capacitation, aliquots of 8–10 × 10⁶ sperm were taken for protein extraction at 180 min of incubation only. Spermatozoa were first washed (600 × g, 10 min) in phosphate-buffered saline in order to remove media-derived protein and the resulting pellet was suspended 1:1 in lysis buffer (62.6 mM Tris, 1 mM sodium orthovanadate, 2% w/v SDS, complete ultra mini EDTA-free protease inhibitor tablet) before being kept at room temperature for 1 h with frequent vortexing. Following this lysis period, samples were then centrifuged (7500 × g, 15 min) and the lysate was retained. The protein concentration of lysates was estimated using Quant-iT Protein Assay (Invitrogen; NSW, Australia) and standardized with Milli-Q water to 1 mg/mL before further dilution with loading buffer (final concentration of 62.5 mM Tris, pH 6.8; 5% (v/v) 2-mercaptoethanol; 2% (v/v) SDS; 10% glycerol (v/v); 0.2% (w/v) bromophenol blue). Samples were then incubated for 5 min at 95°C and then stored at −80°C until required.