| Mean count of neutrophils attached to one melanoma cell by mechanism of frustrated phagocytosis |
---|
Ligand | 20 min. | 30 min. | 40 min. |
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Mannan-BAM | 0.333 | 0.556 | 0.733 |
Mannan free | 0.111 | 0.000 | 0.000 |
f-MLFKK-BAM | 0.589 | 0.311 | 0.360 |
f-MLF free | 0.480 | 0.111 | 0.000 |
PBS (control) | 0.000 | 0.000 | 0.000 |
- B16-F10 melanoma cells were incubated (30 min, 37 °C) with 0.02 mM mannan-BAM or 0.05 mM f-MLFKK-BAM in culture medium and then subsequently washed. Suspension of bone marrow neutrophils (90% purity) primed with GM-CSF + TNF-alpha (+ laminarin in case of mannan-BAM) in culture medium was added to B16-F10 cells. Free ligands were added in a concentration of 0.02 mM (mannan) and 0.05 mM (f-MLFKK). Neutrophils and melanoma cells were incubated at a 2:1 ratio
- The rate of frustrated phagocytosis events was estimated by light microscopy: Frustrated phagocytosis was defined as neutrophil/melanoma cell contact, where neutrophil adhere tightly to B16-F10 cell. Such contact is further characterized by neutrophil flattening and gaining of waning moon shape [5]