Euthanasia and BAL Collection for Cytokine Analysis

Animals were euthanized with Euthasol (pentobarbital sodium/phenytoin sodium) prior to BAL and blood collection. A mid-line dissection through the abdominal cavity up to the thoracic cavity was performed followed by a small incision at the edge of the diaphragm, which resulted in a fatal pneumothorax. Then, a right ventricular puncture for the collection of blood was performed. Blood was collected in a microvette microtube with EDTA (Kent Scientific) and kept on ice and then spun at 2000 rpm. Serum was collected and used for IL-6 determination by ELISA (Thermo Fisher, KMC0062) (37 (link)). Bronchoalveolar lavage (BAL) fluid was obtained through a 20-gauge angiocath ligated into the trachea through a tracheostomy. A total of 1-ml of PBS instilled into the lungs was aspirated three times to collect BAL fluid for cell counts (Cellometer K2; Nexcelom Bioscience), protein quantification (Bradford assay) and IL-6 determination by ELISA.

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Radigan K.A., Nicholson T.T., Welch L.C., Chi M., Amarelle L., Angulo M., Shigemura M., Shigemura A., Runyan C.E., Morales-Nebreda L., Perlman H., Ceco E., Lecuona E., Dada L.A., Misharin A.V., Mutlu G.M., Sznajder J.I, & Budinger G.R. (2018). Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1. Journal of immunology (Baltimore, Md. : 1950), 202(2), 484-493.

Publication 2018

Cellometer K2

Abdominal cavity Animals Assay Blood Bronchoalveolar lavage Bronchoalveolar lavage fluid Diaphragm Dissection Edta Elisa Lungs Pentobarbital sodium Phenytoin sodium Pneumothorax Protein Puncture Right ventricular Serum Thoracic cavity Trachea Tracheostomy

Corresponding Organization : Northwestern University

Other organizations : Universidad de la República, University of Chicago

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Variable analysis

independent variables

Euthanasia with Euthasol (pentobarbital sodium/phenytoin sodium)

dependent variables

IL-6 levels in serum
IL-6 levels in bronchoalveolar lavage (BAL) fluid
Cell counts in BAL fluid
Protein quantification in BAL fluid

control variables

Dissection procedure to obtain blood and BAL samples
Collection of blood in microvette microtube with EDTA
Centrifugation of blood samples at 2000 rpm
Instillation of 1-ml of PBS into the lungs and aspiration 3 times to collect BAL fluid

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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