In vivo assay biochem kit

In brief, a three-step enzymatic digestion method was used. The seminiferous tubules were digested twice with collagenase type I (Worthington) at 200U/ml for 15 min at 33°C in DMEM (Gibco) supplemented with DNase I (10U/ml, Promega). The pellets were further digested with 0.05%Trypsin-EDTA (Gibco) for 5 min and stopped by 2% serum. The resulting cell suspension was filtered through a 40 μm cell strainer (BD). The harvested cells were diluted for Hoechst (5 μg/ml) and PI (2 μg/ml) staining, and then prepared for FACS analysis (LSR II Flow Cytometer, BD) or cell sorting (Aria II Flow Cytometer, BD). Membrane and cytoplasmic proteins were extracted with a membrane and cytoplasmic protein extraction kit (Beyotime Biotechnology). The F-actin/G-actin was isolated by the In Vivo Assay Biochem Kit (Cytoskeleton). The RhoA/Rac1/Cdc42 activities were measured by G-LISA Activation Assay Kit (Cytoskeleton).

The relative abundance of F-actin and G-actin with or without shikonin treatment was determined using an In Vivo Assay Biochem Kit (Cytoskeleton, Inc., Denver, CO). Briefly, the T24 cells were exposed to either DMSO or that containing 0.5 μM shikonin for 6 h. The cells were then lysed in LAS2 buffer and the lysate was centrifuged at 350 × g for 5 min at room temperature. The supernatant was centrifuged at 100,000 × g at 37 °C for 1 h to separate F-actin from G-actin. The pellet containing F-actin was re-suspended in actin de-polymerization buffer on ice for 1 h. Equal volumes of G-actin supernatant and F-actin resuspension were analyzed by Western-blotting and the actin bands were semi-quantified using NIH ImageJ software.