Bovine testes type 1 s

The surface antigens on the cells were analyzed via flow cytometry. Cells encapsulated in the hydrogels were washed twice with DPBS and dissociated using hyaluronidase from bovine testes Type I-s (Sigma Aldrich) before collection by centrifugation at 1,300 rpm for 3 min. The cell pellets were washed twice with DPBS and blocked with 2 % FBS in DPBS solution (FACS buffer). Specific antibodies (diluted 1:100 in FACS buffer) were incubated at 4 °C for 30 min and washed thrice with FACS buffer. Finally, fluorescence was detected using BD Accuri C6 (BD Bioscience, Japan). The percentage of expressed cell surface antigens was calculated for 10,000 gated cell events. The antibodies used in these experiments were anti-CD44 (103011, Biolegend, UK) and anti-SSEA1 (125608, Biolegend).

Sperm were preincubated in a modified Krebs-Ringer bicarbonate solution (TYH) containing 0.75 mM methyl-β-cyclodextrin (MBCD, Sigma-Aldrich, St, Louis, MO, USA) that strongly promotes sperm capacitation by facilitating cholesterol efflux from the plasma membrane of the sperm [3 , 22 (link)]. Modified human tubal fluid (mHTF) with a high concentration of calcium (5.14 mM), which is bicarbonate-buffered solution, was used as the cumulus removal and fertilization mediums [23 (link), 24 (link)]. Potassium simplex optimization medium (KSOM) was used to culture embryos to the blastocyst stage [25 (link)]. Hyaluronidase was purchased from Sigma-Aldrich (400–1000 unit/mg of Hyaluronidase from bovine testes, Type I-S; Sigma-Aldrich). The titer of Hyaluronidase (Lot No. 029K7001V), measured by Sigma-Aldrich, was 801
unit/ml. A stock solution of 1% Hyaluronidase was prepared in mHTF and stored at – 20 C until use.