This video demonstrates an assay for quantifying human norovirus virus-like particles (VLPs) binding to human intestinal bacteria. The bacterial suspensions are mixed with the VLPs and incubated to allow the formation of VLP-bacteria complexes. The complexes are then labeled with fluorophore-tagged VLP-specific antibodies, followed by flow cytometry-based visualization and quantification to identify the percentage of VLP-bound bacteria.
Protocol
1. VLP-bacteria Attachment Assay CAUTION: Human norovirus VLPs are a biosafety level (BSL)-2 hazard, and all work involving VLPs should be performed in a biosafety cabinet. Preparation of the bacterial cultures before the attachment assay should be performed using safety conditions appropriate for the organism. Reagent preparation 1x phosphate-buffered saline (PBS) Prepare 1 L of 10x PBS by dissolvi…
Offenlegungen
The authors have nothing to disclose.
Materials
5ml Polystrene Round-Bottom Tubes with Cell-Strainer Cap
Corning
352235
After antibody staining, sample are transferred into tubes for flow cytometry analysis.
AnaeroPack
Thermo Scientific
R681001
Anaerobic gas pack used for culture of Lactobacillus gasseri
BD FacsDiva software
BD LSR Fortessa flow cytometer
Bovine Serum Albumin
Fisher Bioreagents
BP1605
Used for flow cytometry
Flow Cytometry Stain Buffer (FCSB)
BD Biosciences
554657
Used for flow cytometry
Mouse IgG2b kappa Isotype Control (eBMG2b), PE, eBioscience
Thermo Fisher Scientific
12473281
Isotype control. This antibody is purchased in the conjugated form from the manufacturer.
MRS Powder
BD Biosciences
288130
Used for media preparation and to culture Lactobacillus gasseri.
Norovirus capsid G2 Monoclonal Antibody (L34D)
Thermo Fisher Scientific
MA5-18241
Norovirus GII antibody. This antibody is only available in the unconjugated form and thus must be fluorescently conjugated prior to use in the outlined flow cytometry assays. In this protocol, PE was the chosen fluor, however, other fluorescent molecules can be chosen as best suits the flow cytometer being used by the researcher.
Norovirus GII.4 VLP
Creative Biostructure
CBS-V700
Human norovirus virus like particle, VLPs were generated using the baculovirus system and resuspended in phosphate buffered saline with 10% glycerol. The authors performed independent nanosight tracking analysis to determine the particle concentration of the VLPs. The concentration is approximately 1011 VLPs per milliliter. Based on the protein concentration of the VLPs, approximately 200 particles are added per bacterium in VLP attachment assays.
PBS 10X
Fisher Bioreagents
BP665
Dilute to 1X prior to use.
SiteClick R-PE Antibody Labeling Kit
Thermo Fisher Scientific
S10467
Conjugation kit used for labling of unconjugated antibody.
Sodium Chloride
Fisher Scientific
S271
Used for media preparation
Tryptone
Oxoid
LP0042
Used for media preparation
Tube Revolver
ThermoFisher Scientific
88881001
Used in virus:bacterium attachment assay. Set to max speed (40 rpm).