Rationale Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a human cytokine that plays a major role in the activation and survival of neutrophils, eosinophils, and monocytes. It increases the activation and survival rates of these cells by inhibiting apoptosis and increasing adhesion. Specifically in neutrophils, GM-CSF is known to enhance cell chemotaxis, expression of membrane antigens, and degranulation of lytic enzymes. GM-CSF, when applied topically, has been shown to improve wound healing, particularly in patients with inherit defects in neutrophil function.
Methodology In order to quantify the effects of GM-CSF on neutrophil activation, we monitored their morphology and utilized flow cytometric analysis at various concentrations of the drug. We first isolated white blood cells from whole blood by centrifuging through a 75% percoll gradient. After the target cells were separated, uniform cultures diluted to 1.0 × 106 cells/mL were prepared. These cultures were then subjected to various concentrations of GM-CSF and incubated over various increments of time (1 hour, 2 hours, 24 hours). After the allotted time, further activation was prevented with gluteradehyde. Activation through morphology was then determined by sampling the number of cells with morphological elongation and the presence of pseudopods at the various concentrations of GM-CSF. Flow cytometry was performed to monitor neutrophil activation through CD69 and CD16 expression.
Results Preliminary results indicate that GM-CSF will induce morphological changes in neutrophils that may be monitored by this assay.
Conclusions We hope to eventually correlate these in vitro changes with the effects of topical GM-CSF in wound healing.
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