An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria

Abstract

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

This video demonstrates an in vitro technique to stimulate lymphocytes via non-typeable Haemophilus influenzae (NTHi) and to assess the immune response. Incubating isolated peripheral blood mononuclear cells (PBMCs) with NTHi causes the bacterial cells to stimulate T lymphocytes, leading to cytokine production. Upon inhibiting the secretion of cytokines, immunofluorescence staining is performed to detect the accumulated intracellular cytokines in stimulated T lymphocytes.

Protocol

1. Antigenic preparation NOTE: Three different antigenic preparations can be used to assess the immune response to Hi. These are 1) a subcellular component (typically from the bacterial cell wall); 2) killed and inactivated bacteria; and 3) live bacteria. Determine the use of each antigenic preparation prior to the initiation of any experiments. Subcellular components Obtain subcellular components from sources, including commercial prep…

Representative Results

Figure 1: Cytokine production in lung tissue. Cells are first analyzed for their expression of the leukocyte marker CD45 (A) using flow cytometry. This population is then analyzed further for CD3 expression (B) and CD4/CD8 expression (C). CD3/CD4+ cells are assessed for intracellular cytokine production in control (D) and NTHi-st…

Materials

Ammonium chloride	Sigma Aldrich	213330
Brefeldin	Sigma Aldrich	B6542
CD28	Thermofisher	16-0289-81
CD49d	Thermofisher	534048
Saponin	Sigma Aldrich	8047152