MPO levels in the spleen, as an assessment of neutrophil influx in tissue homogenates, were measured 24 h post-CLP by ELISA according to the manufacturer’s instructions (#EMMPO; Thermo Fisher Scientific, Waltham, Mass). Similar to cytokine analysis, the 24 h time point was selected as myeloid cells are known to respond robustly and rapidly to septic stimuli within this time frame. To measure esterase levels, spleen sections were stained with naphthol AS-D chloroacetate esterase (Sigma-Aldrich, St. Louis, Mo) according to the manufacturer’s instructions. The images were collected with the Nikon Eclipse 80i microscope using a ×20 and ×40 objective and Spot RT3 camera. Slides were randomly screened and blindly evaluated with three to six images acquired per specimen (42 (link)–44 (link)).
Protocol detail
Quantifying Neutrophil Influx in Sepsis
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Chloroacetate esterase
Cytokine
Elisa
Esterase
Frame
Microscope
Myeloid cells
Naphthol
Neutrophil
Septic
Spleen
Tissue
Corresponding Organization :
Other organizations : Lifespan, Rhode Island Hospital, Washington University in St. Louis
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Variable analysis
independent variables
- Time post-CLP (24 h)
- MPO levels in the spleen (as an assessment of neutrophil influx in tissue homogenates)
- Esterase levels in spleen sections
- ELISA assay according to the manufacturer's instructions
- Naphthol AS-D chloroacetate esterase staining of spleen sections according to the manufacturer's instructions
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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