Quantification of Inflammatory Mediators in Infected Human Tissue-Derived Cells

Abstract

Source: Dousha, L. et al., Assessing Respiratory Immune Responses to Haemophilus Influenzae. J. Vis. Exp. (2021)

This video demonstrates the quantification of inflammatory mediators during infection by incubating human lung-tissue-derived immune cells with Haemophilus influenzae. Flow cytometry is used to measure cytokine fluorescence in labeled lymphocytes, correlating with the production of pro-inflammatory cytokines in response to bacterial infection.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Antigenic preparation

1. Live bacteria
   1. First, characterize live bacteria. Obtain the bacteria from the appropriate sample (e.g., sputum or bronchoscopy). Confirm the strain as Haemophilus influenzae using an appropriate microbiology laboratory. Perform typing of the H. influenzae samples to confirm they are nontypeable (by a specialist microbio....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium chloride	Sigma Aldrich	213330
Brefeldin	Sigma Aldrich	B6542
Filcon sterile nylon mesh	Becton Dickinson	340606
Gelatin substrate, Enzchek	Molecular probes	E12055
MACS mix tube rotater	Miltenyi Biotec	130-090-753
Medimachine	Becton Dickinson		Catalogue number not available
Saponin	Sigma Aldrich	8047152