Bca protein assay reagent kit

Proteins were extracted from cells at appropriate time points after drug treatment using RIPA buffer [50 mM Tris–HCl (pH 8), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Protein concentrations were measured using a BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, United States). Proteins were separated on SDS polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and probed with specific antibodies. Immunoreactive polypeptides were detected with chemiluminescence using ImmunoStar LD (Wako, Osaka, Japan).

The TP level in BAL fluid was measured using a BCA Protein Assay Reagent Kit (Thermo Fisher Scientific). LDH activity in BAL fluid was measured using the LDH-Cytotoxic Test (Wako Chemicals).

Cells were treated with 10 ng/mL cfDNA, 50 nmol/mL chloroquine, cfDNA plus chloroquine, and DMSO as a control for 24 h. The cells were then harvested quickly in lysis buffer and clarified by centrifugation. A BCA protein assay reagent kit (Thermo Scientific, USA) was used to measure the protein concentration. Then, western blotting was done. We also used DNase I to digest DNA to further verify the effect of cfDNA. First, agarose gel electrophoresis (AGE) was performed to prove that the DNA had been digested by DNase I. DNase I was added into the supernatant of T47-D and MDA-MB-231 cell, and then cfDNA was extracted. ALU gene and β-actin genes were amplified by PCR as controls. AGE was used to detect the amount of DNA. After that, cells were treated with 10 ng/mL cfDNA, 30 U/mL DNase I, cfDNA plus DNase I, and DMSO as control for 24 h.

Hippocampus mitochondria were isolated as described previously [15 (link)]. Following the passive avoidance test, mice were sacrificed by decapitation, the brains were removed, and the hippocampus was dissected on ice. Hippocampus from four mice in one group was pooled to obtain sufficient amount of protein for each group for biochemical analysis. Tissues were homogenized in an ice-cold isolation buffer containing 230 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl (pH 7.4) with a weight/volume ratio of 1 : 10. The homogenate was centrifuged at 700 g for 10 min, followed by centrifugation of the supernatant at 8,000 g for 10 min to obtain a mitochondrial pellet. The pellet was washed twice and resuspended in isolation medium containing 15% Percoll and layered on preformed Percoll gradients (40 and 23%). The gradient was spun at 30,700 g for 10 min, and the 23/40 interphase layer, containing the mitochondria, was collected. The mitochondria fraction was diluted with isolation buffer and centrifuged at 16, 700 g for 12 min. The pellet was then washed with isolation buffer containing bovine serum albumin (BSA) and centrifuged at 9,800 g for 10 min. The pellet containing purified mitochondria was resuspended in 100 μL of isolation buffer without EDTA. Mitochondrial protein content was assayed using a BCA protein assay reagent kit (Thermo Scientific, USA).

RNA was isolated (including DNase I digestion) using the RNAeasy Mini Kit (Qiagen, Hilden, Germany) according to the manual.
Proteins were extracted by RIPA buffer containing 10% protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations were assessed by the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Dreieich, Germany).