Source: Chua, J., et al. Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens. J. Vis. Exp. (2020)
This video demonstrates an opsono-adherence assay for examining the interaction between opsonized bacteria and immune cells. Fluorescently labeled bacteria, treated with test serum containing anti-capsule antibodies, are incubated with macrophages. After staining the macrophages with fluorescence, the attached bacteria on the macrophage surface are observed under a microscope for visualization.
1. Bacterial Culture and Preparations
NOTE: The bacterial species used in this protocol is an encapsulated virulent strain, B. anthracis Ames. All manipulations with this species require appropriate biosafety and security clearances and must be performed in a Class II or Class III biological safety cabinet located in a BSL-3 laboratory. Follow institutional operating procedures for BSL-3 work and for the use of personal protective equipment when handling bacteria.
2. Negative staining and light microscopy to observe encapsulation and bacilli chains
3. FITC-labeling of bacteria
4. Bacterial Opsonization
5.Adherence Assay and Fluorescent Labeling of Mammalian cells
6. Microscopy
NOTE: In general, a high-throughput automated fluorescence microscope will facilitate imaging for the OAA. If an automated system is not available, fluorescent images of adherent bacilli can be taken manually. In this study, adherent bacilli and cell monolayers were imaged using the Zeiss 700 laser scanning microscopy system with specialized equipment; our system was composed of the Zeiss Axio Observer Z1 inverted microscope equipped with an automated stage, the Definite Focus module, 40 x NA 0.6 objective, Axio Cam HRc camera and Zen 2012 (Blue Edition) software. The images acquired are widefield images, not confocal images. The following protocol is intended as a basic microscope setup that is applicable to a variety of automated microscopy systems.
The authors have nothing to disclose.
0.20 µm syringe filter (25mm, regenerated cellulose) | Corning, Corning, NY | 431222 | |
10 mL syringe (Luer-Lock tip) | BD, Franklin Lakes, NJ | 302995 | |
15µ 96 well black plates (plate #1 for imaging) | In Vitro Scientific, Sunnyvale, CA | P96-1-N | |
16% paraformaldehyde | Electron Microscopy Science, Hatfield, PA | 15710 | |
75 cm sq. tissue culture treated flask | Corning, Corning, NY | 430641 | |
Agar (powder) | Sigma-Aldrich, St. Louis, MO | A1296 | |
Baby Rabbit Complement | Cedarlane Labs, Burlington, NC | CL3441 | |
Bacto Yeast Extract | BD, Sparks, MD | 288620 | |
BBL Brain Heart Infusion (BHI) | BD, Sparks, MD | 211059 | |
Blood Agar (TSA with Sheep Blood) plates | Remel, Lenexa, KS | R01198 | |
Cell scraper | Sarstedt, Newton, NC | 83.183 | |
Costar 96 well cell culture plates (plates #2 & 3 for dilutions) | Corning, Corning, NY | 3596 | |
Cover glass | Electron Microscopy Science, Hatfield, PA | 72200-10 | |
Difco Nutrient Broth | BD, Sparks, MD | 234000 | |
Dulbecco's Modified Eagle Medium (DMEM), high glucose | Gibco, Thermo Fisher Scientific, Waltham, MA | 11965-092 | Contains 4500 mg/L glucose, 4 mM L-glutamine, Phenol Red |
EVOS FL Auto Cell Imaging System (fluorescence microscope) | Life Technologies, Thermo Fisher Scientific, Waltham, MA | AMAFD1000 | |
Fetal Bovine Serum | Hyclone, GE Healthcare Life Sciences, South Logan, UT | SH30071.03 | Not gamma irradiated, not heat inactivated |
Fluorescein isothiocyanate | Invitrogen, Thermo Fisher Scientific, Waltham, MA | F143 | |
HCS Cell Mask Orange Cell Stain | Invitrogen, Thermo Fisher Scientific, Waltham, MA | H32713 | |
hemocytometer (Improved Neubauer) | Hausser Scientific, Horsham, PA | 3900 | |
India Ink solution | BD, Sparks, MD | 261194 | |
L- glutamine (200 mM) | Gibco, Thermo Fisher Scientific, Waltham MA | 25030081 | Supplement medium with additional 2mM L-glutamine |
Nikon Eclipse TE2000-U (inverted compound microscope) | Nikon Instruments, Melville, NY | TE2000 | |
PBS without Calcium or Magnesium | Lonza, Walkersville, MD | 17-516F | |
Penicillin-Streptomycin Solution, 100x | Hyclone, GE Healthcare Life Sciences, South Logan, UT | SV30010 | |
Petri dishes (100 x 15 mm) | Falcon, Corning, Durham, NC | 351029 | For agar plates |
RAW 264.7 macrophage cell line (Tib47) | ATCC, Manassas, VA | ATCC TIB-71 | |
Slides | VWR, Radnor, PA | 16004-422 | |
Sodium Bicarbonate | Sigma-Aldrich, St. Louis, MO | S5761 | |
Trypan Blue Solution (0.4%) | Sigma-Aldrich, St. Louis, MO | T8154 | |
Zeiss 700 Laser Scanning Microscopy (confocal microscope) | Carl Zeiss Microimaging, Thornwood, NY | 4.109E+15 |