2.6. Neutral Red Uptake Assay for Phagocytic Activity

The phagocytic activity of RAW 264.7 cells was investigated using a neutral red uptake assay, as previously described [5]. Briefly, RAW 264.7 cells (1 × 10⁵ cells/well) were added to 96-well plates and cultivated at 37 °C with 5% CO₂. After overnight growth, the cells were treated with CFS (5 mg/mL) for 24 h. Thereafter, RAW 264.7 cells were washed three times with PBS, 100 μL of neutral red solution (0.01%) was added to each well, and the cells were incubated for 2 h. After washing three times, each well was treated with a cell lysis solution (acetic acid: ethanol = 1:1), and the plate was incubated at 37 °C for 1 h. Absorbance was measured at 540 nm using a microplate reader.