High-Throughput Imaging of Intracellular Brucella abortus Infection in Host Epithelial Cells

Abstract

Source: Casanova, A., et al. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells. J. Vis. Exp. (2016)

This video demonstrates high-throughput imaging of Brucella abortus infection in epithelial cells with siRNA-mediated STING knockdown to investigate the role of cytosolic DNA sensing in intracellular bacterial replication and host innate immune response.

Protocol

1. Infection and Fixation

1. One day prior to infection, inoculate the determined amount of B. abortus starter culture in 50 ml TSB/Km (Tryptic Soy Broth containing 50 µg/ml kanamycin) medium in a 250 ml screw cap bottle. Seal the bottle with Parafilm and grow bacteria overnight at 37 °C and 100 rpm on an orbital shaker to OD₆₀₀ = 0.8-1.1.
2. Measure OD₆₀₀ of the bacterial culture and prepare the infection medium by diluting the bacterial culture in DMEM (Dulbecco's Modified Eagle Medium) /10% FCS (Fetal calf serum) to achieve the desired multiplicity of infection (MOI).
   NOTE: To obtain an infection....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptic Soy Broth (TSB)	Fluka	22092
Kanamycin sulfate	Sigma-Aldrich	60615
250 ml screw cap bottle	Corning	8396
DMEM	Sigma-Aldrich	D5796
Fetal Calf Serum (FCS)	Gibco	10270	Heat-inactivated
Greiner CELLSTAR 384-well plate	Sigma-Aldrich	M2062
Peelable aluminum foil	Costar	6570
Reagent dispenser: "Multidrop 384 Reagent Dispenser"	Thermo Scientific	5840150	Alternative reagent dispenser can be used.
Transfection reagent: "Lipofectamine RNAiMAX	Invitrogen	13778-150
Automated plate washer: "Plate washer ELx50-16"	BioTek	ELX5016	This plate washer contains a 16-channel manifold suitable for 384-well plates. It fits into a biosafety cabinet and has a lid covering the plate during washing which reduces the risk of aerosol production. Alternative plate washers with similar features could be used.
Gentamicin	Sigma-Aldrich	G1397
PBS	Gibco	20012
Paraformaldehyde	Sigma-Aldrich	P6148	Dissolve in 0.2 M HEPES buffer, pH 7.4. Store at -20 °C and thaw freshly the day before use. Caution, PFA is toxic by inhalation, in contact with skin and if swallowed.
HEPES	Sigma-Aldrich	H3375
Triton X-100	Sigma-Aldrich	T9284
DAPI	Roche	10236276001
Kif11 siRNA	Dharmacon	L-003317-00
ARPC3 siRNA	Dharmacon	L-005284-00
Brucella abortus 2308
HeLa CCL-2	ATCC	CCL-2
ImageXpress Micro	Molecular Devices	IXM IMAGING MSCOPE	Automated cellular imaging microscope equipped with a precision motorized Z-stage. Alternative systems for automated microscopy and alternative components for hard- and software specified below can be employed.