An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria
Published: September 29, 2023
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…
Disclosures
The authors have nothing to disclose.
Materials
| Ammonium chloride | Sigma Aldrich | 213330 | |
| Brefeldin | Sigma Aldrich | B6542 | |
| CD28 | Thermofisher | 16-0289-81 | |
| CD49d | Thermofisher | 534048 | |
| Saponin | Sigma Aldrich | 8047152 |
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Cite This Article
An In Vitro Technique to Stimulate Lymphocytes via Pathogenic Bacteria. J. Vis. Exp. (Pending Publication), e21620, doi: (2023).