Figure 3.

MC extracellular trap (MCET) induction and phagocytosis of Staphylococcus aureus bioparticles. (A) MCETs were visualized without fixation using the live/dead viability/cytotoxicity kit (Invitrogen) for mammalian cells. Significantly more MCETs are found after 24 h hypoxia versus normoxia, but not after 3 h. Results are shown from the analysis of n = 3 independent experiments, each with four individual images. (B) Representative fluorescence micrograph of MCET induction (red: dead cells/MCETs in a fiber like structure; green living cells). (C) Mast cells (MCs) (2 × 10⁶ cells/ml) were preincubated 3 or 24 h under hypoxia (37°C, 1% O₂, 5% CO₂) or normoxia (37°C, 21% O₂, 5% CO₂). Then phycoerythrin (PE)-labeled S. aureus (Wood strain, bioparticles; Sigma) at an MOI of 60 was incubated with MCs for 30 min under the respective oxygen condition. CTR represents uninfected control. The cells were washed with PBS and centrifuged to remove non-phagocytosed bacteria. PE-fluorescence was measured using a Beckman Coulter EPICS XL flow cytometer. The red fluorescence intensity per MC (% gated) was recorded and represents the mean relative phagocytosis of PE-labeled S. aureus per MC of n = 3 independent experiments.