Fig 2: Accutase cleaved surface FasL proteins. (a,b) RAW264.7 cells were incubated with either accutase or EDTA cell detachment buffer for 30 min. The supernatants and lysates in both groups were collected and concentrated. The FasL proteins in the supernatant and lysates were analyzed by western blot. Specific antibodies against the extracellular domain of FasL were used for protein detection. (b) RAW264.7 cells were incubated with either accutase or EDTA cell detachment buffer for 30 min, followed by immunofluorescence staining. The white arrows indicate the FasL proteins around cell edges.

Fig 3: The effects of different cell detachment buffers on surface protein expression in RAW264.7 macrophages. (a,c) The surface expression of Fas ligand (FasL) and Fas receptor was detected by flow cytometry in RAW264.7 murine macrophages treated with either EDTA or accutase for 10 min. (b,d) Statistical analysis revealed a significant difference in surface FasL expression between the EDTA and accutase treatment groups. (e,f) Neither EDTA nor accutase buffer incubation affected surface F4/80 proteins in RAW264.7 cells (p = 0.2). (G,H) The EDTA cell detachment buffer had minor effects on cell surface FasL expression when applied for 30 min compared to those of the cell scraping method. Cells incubated with the accutase cell detachment buffer for either 10 or 30 min exhibited significantly decreased surface FasL expression. (***p < 0.001, one-way ANOVA). MFI mean fluorescence intensity.

Fig 4: Recovery of surface proteins after treatment with accutase detachment buffer. RAW264.7 cells were incubated in accutase buffer for 30 min and then in complete medium for the indicated time amounts of time. Subsequently, the cells were harvested with EDTA detachment buffer for the analysis of surface proteins. (a–d) Significant reductions in the expression levels of the surface FasL and Fas receptor proteins were observed after recovery for 2 h in complete medium. After recovery in complete medium for 7 to 20 h, the levels of the surface FasL and Fas receptor proteins recovered gradually (p < 0.05). (e,f) No significant differences in surface F4/80 proteins were observed after treatment with accutase and recovery for the indicated time amounts of time (p = 0.33). MFI: Mean fluorescence intensity.