Effects of Hydrogels on RAW 264.7 Cells Polarization, Inflammatory Response, and Cytokine Expression
This protocol is extracted from research article:
ROS-Eliminating Carboxymethyl Chitosan Hydrogel to Enhance Burn Wound-Healing Efficacy
Front Pharmacol, Jun 14, 2021; DOI: 10.3389/fphar.2021.679580

To analyze the effects of Cur@CMCTS-Tk hydrogel on macrophages phenotype switch and inflammatory response, a flow cytometer (FCM) and a Western blot analysis were applied. To stimulate the ROS microenvironment, LPS (100 ng/ml) was chosen and co-cultured with Cur@CMCTS-Tk hydrogel. At last, 1.0 × 105 RAW 264.7 cells were cultured with CMCTS-Tk or Cur@CMCTS-Tk hydrogel and LPS condition.

Western blot analysis: Fibroblasts were rinsed in phosphate buffer saline (PBS) and mixed with a radioimmunoprecipitation assay (RIPA) buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (PMSF). The protein was electrophoretically resolved (120 V) on a 12% SDS-polyacrylamide gel and transferred (350 V) to PVDF membrane for 90 min and incubated with 5% skim milk. Afterward, the PVDF membrane was incubated overnight with primary antibodies at 4°C and washed with TBST thrice, for 10 min each time. Next, the membrane was blotted with peroxidase-conjugated secondary antibodies and washed the same number of times as in the previous step with TBST. Visualization of proteins was performed by the chemiluminescent signal following the instructions of the manufacturer. Primary antibodies for TNF-α (ab255275) and IL-10 (ab189392) monoclonal antibodies were used.

FCM analysis: After incubation for 48 h, 10% mouse serum was used to block the RAW 264.7 cells for 30 min. The cells were then incubated in the mixed solution combining rabbit CD86 (ab242142) and CD206 (ab223961) monoclonal antibody (dissolved in PBS) with 0.05% proclin300 and 1% BSA for at 4°C 30 min. After washing with PBS thrice, the RAW 264.7 cells were placed in PBS and analyzed by flow cytometry (Beckman Coulter, California, United States). Besides, the inflammation-associated protein of TNF-α and IL-10 was analyzed by using a flow cytometer which is incubated with rabbit TNF-α (ab255275) and IL-10 (ab215975) monoclonal antibody.

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