Analyzing the Effects of Cigarette Smoke on Antimicrobial Defense in Lung Epithelial Cells

Özet

Source: Li, T., et al. Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells. J. Vis. Exp. (2020).

This video demonstrates how cigarette smoke extract weakens antimicrobial defense in lung epithelial cells by reducing AMP production, leading to increased bacterial invasion. The procedure measures intracellular bacterial load to assess the impact of smoke exposure on host-pathogen interactions.

Protokol

1. Human lung epithelial cell culture and cigarette smoke extract (CSE) treatment

1. Culture human BEAS-2B cells in HITES medium (500 mL of DMEM/F12, 2.5 mg insulin, 2.5 mg transferrin, 2.5 mg sodium selenite, 2.5 mg transferrin, 10 μM hydrocortisone, 10 μM β-estradiol, 10 mM HEPES, and 2 mM L-glutamine) supplemented with 10% fetal bovine serum (FBS) as previously described24.
2. Culture human primary small airway epithelial cells (HSAEC) in the airway epithelial cell culture medium (500 mL of Airway Cell Basal Medium, 500 µg/mL HSA, 0.6 µM linoleic acid, 0.6 µg/mL....

Malzemeler

Bu makalede kullanılan malzemelerin listesi

Ad	Şirket	Katalog numarası	Yorumlar
50mL syringe	BD Biosciences
Airway epithelial cell basal medium	ATCC	PCS-300-030
Bacteria shaker	ThermoFisher Scientific
Bronchial epithelial cell growth kit	ATCC	PCS-300-040
Cell Counter	Bio-Rad
CFX96 Real-Time PCR System	Bio-Rad
High-Capacity RNA-to-DNA KIT	ThermoFisher Scientific	4387406
HITES medium	ATCC	ATCC 30-2004
Human BEAS-2B cell	ATCC	ATCC CRL-9609
Human primary small airway epithelial cells	ATCC	ATCC PCS-300-030
OD reader	USA Scientific
Pseudomonas aeruginosa	ATCC	ATCC 47085	PAO1-LAC
Pseudomonas fluorescens Migula	ATCC	ATCC 27853	P.aeruginosa GFP
Research-grade cigarettes (3R4F)	University of Kentucky	TP-7-VA
Tryptic Soy Broth	BD Biosciences