An Opsono-Adherence Assay for Assessing the Opsonization of Encapsulated Bacteria by Anti-Capsule Antibodies

Published: January 31, 2024

Abstract

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.

Protocol

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 handlin…

Declarações

The authors have nothing to disclose.

Materials

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

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An Opsono-Adherence Assay for Assessing the Opsonization of Encapsulated Bacteria by Anti-Capsule Antibodies. J. Vis. Exp. (Pending Publication), e21879, doi: (2024).

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