Protocol to infect differentiated human primary bronchial epithelial cells with live mycobacteria and determine intracellular load

STAR Protocols, vol. 6, pp. 103871

Abstract

Lung epithelial cells present the first line of defense against pathogens like Mycobacterium tuberculosis and other related species. Studying their responses is instrumental to understand early infection stages of tuberculosis. We present a protocol to study the infection potential of mycobacteria in differentiated primary human bronchial epithelial cell cultures. We describe mycobacterial and epithelial cell culture techniques. We then detail how to perform infections in epithelial cells and determine intracellular bacterial load using flow cytometry and colony-forming unit assays. For complete details on the use and execution of this protocol, please refer to Barclay et al. 1 , 2

Authors 7

1. Amy M Barclay corresponding Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center (LUMC), Leiden 2333ZA, the Netherlands. Electronic address: A.M.de_Waal@lumc.nl

2. Kimberley V. Walburg corresponding Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center (LUMC), Leiden 2333ZA, the Netherlands. Electronic address: K.V.Walburg@lumc.nl

3. Dennis K. Ninaber Aachen PulmoScience Lab Department of Pulmonology, LUMC

   Leiden University Medical Center

   Affiliation as printed

   PulmoScience Lab, Department of Pulmonology, LUMC, Leiden 2333ZA, the Netherlands

4. Tom H. M. Ottenhoff Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center (LUMC), Leiden 2333ZA, the Netherlands

5. Pieter Sicco Hiemstra Aachen PulmoScience Lab Department of Pulmonology, LUMC

   Leiden University Medical Center

   Affiliation as printed

   PulmoScience Lab, Department of Pulmonology, LUMC, Leiden 2333ZA, the Netherlands

6. Anne M. van der Does Aachen PulmoScience Lab Department of Pulmonology, LUMC

   Leiden University Medical Center

   Affiliation as printed

   PulmoScience Lab, Department of Pulmonology, LUMC, Leiden 2333ZA, the Netherlands

7. Simone A. Joosten corresponding Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Center for Infectious Diseases (LUCID), Leiden University Medical Center (LUMC), Leiden 2333ZA, the Netherlands. Electronic address: S.A.Joosten@lumc.nl