The Use of a Flow Cytometry-Based Cytokine Analysis for Bronchoalveolar Lavage with Confirmation by Real-Time RT-PCR in a Mouse Model of Pulmonary Inflammation

Details

• Personal Author:
  Antonini JM ; Erdely AD ; Young S ; Zeidler-Erdely PC
• Description:
  Bronchoalveolar lavage (BAL) provides a valuable tool for insight into the lung inflammatory state following a toxic or antigenic exposure. Toxicological injury evaluation routinely uses a concentrated first fraction BAL (<600 µl) to accurately measure various lung inflammatory parameters in mice. However, 100-250 µl of this BAL fraction is typically used in most conventional assays limiting the researcher to only a few measurements. Recently, flow-cytometry based cytometric bead arrays (CBA) have been developed to simultaneously determine concentrations of multiple analytes in a single 25-50 µl sample saving both cost and time. In this confirmatory study, we used a mouse inflammation CBA kit to analyze IL-6, IL-10, IL-12p70, MCP-1, IFN-gamma, and TNF-alpha protein in BAL fluid obtained from A/J or C57BL/6J mice exposed to welding fume. Since inflammatory mediators are usually accompanied by both an increase in gene expression and protein, real-time RT-PCR was done on isolated RNA from whole lung homogenates to validate the findings. It was found that a limitation of the CBA kit was sensitivity. In particular, samples with low concentrations of an analyte were often reported as extrapolated below sensitivity or zero thus limiting statistical analysis. These findings made validation by some other method necessary. Real-time RT-PCR strongly supported the findings of the CBA kit by displaying similar trends in the mouse lung inflammatory response caused by welding fume exposure. Therefore, the mouse inflammation CBA kit was an acceptable method for measuring cytokines in studies involving limited sample size and represents a cost efficient screening method for pulmonary inflammation. Also, it is recommended that another method such as RT-PCR or ELISA be performed if increased sensitivity is required. [Description provided by NIOSH]
• Subjects:
  Animals Energy Metabolism Laboratories Lung Lung Diseases Occupational Health Respiratory Function Tests Respiratory System Respiratory Tract Diseases Safety
• Keywords:
  Alveolar-cells Animal-studies Biodynamics Laboratory-animals Lung-cells Lung-function Lung-irritants Metabolism Models Pulmonary-disorders Pulmonary-function-tests Pulmonary-system-disorders

  More +
• ISSN:
  1096-6080
• Document Type:
  Text
• Genre:
  Abstract or Summary
• Place as Subject:
  OSHA Region 3 ; West Virginia
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Division:
  HELD - Health Effects Laboratory Division
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  102
• Volume:
  96
• Issue:
  1
• NIOSHTIC Number:
  nn:20031907
• Citation:
  Toxicologist 2007 Mar; 96(1):103
• Federal Fiscal Year:
  2007
• Peer Reviewed:
  False
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:8bbf98a3c27d19fdbd7cb5172bd005dd4c52267cf869b094b4273433a968904473cc56da91ab1e3e851156ce2300899befc7bb14c461dbf8029caa414f5aa027
• Download URL:
  https://stacks.cdc.gov/view/cdc/186018/cdc_186018_DS1.pdf
• File Type:
  [PDF - 518.16 KB ]