Microbca assay kit

To examine FN persistence time, composite hydrogels fabricated through physical mixing and covalent grafting were made into uniformly sized blocks and placed in 6-well plates. 5 wt% HAMA solution with fibronectin (50 μg/ml) was used in the physical mixing group, and 5 wt% FN-HAMA (75% grafting ratio) solution was used in the covalent grafting group. The solution in the supernatant was collected after 24, 48, 72, and 96 h of incubation in PBS to assess the retention of FN. Quantification was performed using the bicinchoninic acid colorimetric assay (MicroBCA Assay Kit, Beyotime Biotechnology). Briefly, standards and samples mixed with working reagents are loaded into a 96-well microplate. The microtiter plate was then sealed and incubated for 2 h at 37 °C. Following incubation, the microtiter plate was allowed to cool at RT for 20 min, protected from light. Measure the absorbance at 562 nm using a plate reader (BIOTEK).

Icaritin (>98% purity) was purchased from Shanghai U-sea Biotech Co. Ltd. modified Eagle's medium of alpha (α-MEM) and fetal bovine serum (FBS) were both obtained from Hyclone (Hyclone; GE Healthcare Life Sciences); The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. Dimethyl sulfoxide (DMSO), trypsin and TRIzol^® reagent were purchased from Gibco; Thermo Fisher Scientific, Inc.; the alkaline phosphatase (ALP) activity kit, enhanced chemiluminescent detection reagent and micro-BCA assay kit were obtained from Beyotime Institute of Biotechnology. DKK-1 and ICI182780 were purchased from R&D Systems, Inc. The RNA extraction kit was purchased from Takara Bio, Inc. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) primers were obtained from Invitrogen; Thermo Fisher Scientific, Inc. The antibodies used were obtained from Cell Signaling Technology, Inc., unless otherwise indicated. Other reagents used in the experiment were purchased from Sigma-Aldrich; Merck KGaA. Icaritin was dissolved in DMSO and the final concentration of DMSO was 0.05% (v/v). ICI 182780 was dissolved in DMSO and stored at 4°C. Cells were pretreated with ICI 182780 (1 µM) for 30 min at 37°C, followed by icaritin treatment.