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. 2021 Sep 18;22(18):10106. doi: 10.3390/ijms221810106

Figure 1.

Figure 1

Dual stimulation of neutrophils is required for efficient degranulation. Neutrophils were cultured for 2 h in the absence or presence of different stimuli used at fixed concentrations (GM-CSF: 50 U/mL, LPS: 10 ng/mL, fMLF: 1 µM, and/or TNF: 1 ng/mL), (A,B,D–F) or increasing concentrations (C). (A) Flow cytometry plot demonstrating gating strategy to determine neutrophil degranulation: CD16-CD63+, or azurophilic degranulation: CD63+ (CD16-CD63+ and CD16+CD63+). (B) Flow cytometry plot demonstrating that neutrophils expressing CD16-CD63+ are consistently high in degranulation of specific and gelatinase granules (CD66b). (C) Effects of increasing concentrations of the different stimuli on neutrophil degranulation displayed as percentages of CD16-CD63+ neutrophils. (D) Effect of single and double stimulation on degranulation as measured by percentages of CD16- neutrophils. (E) Effect of single and double stimulation on degranulation as measured by percentages of CD63+ neutrophils. (F) Effect of single and double stimulation on specific and gelatinase granule degranulation as measured by changes in mean fluorescent intensity (MFI) of CD66b, expressed in fold change compared to unstimulated. Data are representative of 6 (C) and 25 (D,E,F) independent experiments, and are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The p-values were calculated using a paired t-test (C) and Friedman test with Dunn’s correction.