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

Samenvatting

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 rate of 5-10% in HeLa cells, an MOI of 10,000 is used. A MOI titration curve should be performed for each cell type to obtain optimal infection rates.
3. Exchange the transfection medium of the 384-well plate with 50 µl of the infection medium using an automated plate washer.
4. Centrifuge the 384-well plate at 400 x g and 4 °C for 20 min.
   NOTE: This helps to synchronize the infection process.
5. Seal the plate with Parafilm and incubate it on a pre-warmed aluminum plate in a 37 °C humid incubator with 5% CO₂ for 4 hr.
6. Wash the cells with DMEM/10% FCS containing 100 µg/ml Gm (gentamicin) to inactivate extracellular bacteria using the automated plate washer (use 200 µl/well overflow wash).
   NOTE: During an overflow wash, the automated plate washer simultaneously dispenses and aspirates medium.
7. For the entry assay, wash cells a second time with DMEM/10% FCS containing 100 µg/ml Gm and 100 ng/ml aTc using the automated plate washer (use 200 µl/well overflow wash).
8. Seal the plate with Parafilm and return it to a pre-warmed aluminum plate in the 37 °C humid incubator with 5% CO₂ for another 40 hr or 4 hr, for the endpoint assay and the entry assay, respectively.
9. For fixation, wash the wells with PBS (phosphate-buffered saline) using the automated plate washer (use 200 µl/well overflow wash).
10. Exchange PBS with 50 µl 3.7% paraformaldehyde (PFA) in 0.2 M HEPES at pH 7.4 using the automated plate washer.
    NOTE: Caution: PFA is toxic by inhalation, in contact with skin, and if swallowed.
11. Incubate for 20 min at room temperature.
12. Exchange the fixation medium with 50 µl PBS using the automated plate washer. 
    NOTE: The samples are now ready to be taken out of the BSL3 if needed.
    CAUTION: Removal of any sample from a BSL3 (Biosafety Level 3) facility is subject to risk assessment and validation of the procedure and depends on the applicable regulations for biosafety.

2. Staining

1. Wash cells twice with 50 µl PBS.
2. Permeabilize cells in 50 µl 0.1% Triton-X-100 in PBS for 10 min.
3. Wash cells three times with PBS. Add 20 µl of PBS containing 1 µg/ml DAPI (4',6-diamidino-2-phenylindole) and incubate for 30 min at room temperature.
4. Wash cells three times in PBS and protect the staining from light with aluminum foil.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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.