Pierce bovine serum albumin standard pre diluted set

LDL was isolated from human plasma using KBr-density gradient as previously described (Havel et al., 1955 (link)). Briefly, plasma was isolated from blood by centrifugation at 2,000 × g, 4°C for 10 min. Plasma density was adjusted to 1.100 g/ml using specific gravity KBr Solution A (density at 1.006 g/ml) and centrifuged at 17,136 × g, 4°C for 18 h using a 70.1 Ti fixed-angle titanium rotor (Beckman Coulter, 342184, Brea, CA) and Optma LE-8OK Ultracentrifuge (Beckman Coulter, 8043-30-1192). The supernatant fraction containing very-low-density lipoprotein (VLDL) was discarded. Solution density was adjusted using specific gravity KBr Solution B (density at 1,182 g/ml) and centrifuged at 17,136 × g, 4°C for 24 h. The supernatant fraction (orange ring) containing low-density lipoprotein (LDL) was isolated and further dialyzed in 0.85% (w/v) NaCl at 4°C for 24 h. LDL was incubated with 10 μM CuSO₄ at 37°C for 24 h. Oxidized LDL (ox-LDL) was dialyzed in 0.85% (w/v) NaCl at 4°C for 24 h. Protein content was determined using Pierce^TM 660 nm Protein Assay reagent (Thermo Fisher Scientific, 22660) and Pierce^TM Bovine Serum Albumin Standard Pre-diluted Set (Thermo Fisher Scientific, 23208) as standards.

BMDM and PM were seeded at 300,000 cells/well in a 24-well plate (Nest Biotechnology, 702001). Interleukin-1β was quantified in the culture medium using the Mouse Il-1β Uncoated ELISA kit (Thermo Fisher Scientific, 88-7013) according to the manufacturer’s protocol. For normalization, adhered cells were harvested and incubated with 100 µL RIPA buffer (Sigma-Aldrich, R0278) containing cOmplete^TM Protease Inhibitor Cocktail (Roche, 11697498001) for 15 min at 4°C. The cellular lysate was centrifuged at 18,000 × g, 4°C for 15 min. The protein content was determined in the supernatant using Pierce^TM 660 nm Protein Assay reagent (Thermo Fisher Scientific, 22660, Rockford, IL) and Pierce^TM Bovine Serum Albumin Standard Pre-Diluted Set (Thermo Fisher Scientific, 23208) as standards. Absorbance was measured in a SpectraMax M3 spectrophotometer (Molecular Devices) at the wavelength of 660 nm. Results were expressed as interleukin-1β amount by total cell protein.

Three of each cell-seeded mineralized collagen and mineralized collagen–amnion scaffold were lysed for protein at Days 0, 4, 7, 14 and 28. To extract protein, a solution of phosphate inhibitor cocktails (Sigma-Aldrich) and an RIPA lysis buffer were used [42 (link)]. To evaluate the protein concentration, a Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific) and a Pierce^TM Bovine Serum Albumin Standard Pre-Diluted Set (Thermo Fisher Scientific) were used. Analysis of protein activity was quantified with a Western Blot assay, loading 5 µg of protein lysate in each lane and using a SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) or a SuperSignal West Pico PLUS Sensitivity Substrate (Thermo Fisher Scientific) to visualize bands. Images were captured with an Image Quant LAS 4010 machine (GE Healthcare Life Sciences, Little Chalfont, UK). Primary and secondary antibodies are listed in Table 1, and β-actin was used as a loading control.