Source: Bäckström, B. T. A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis. J. Vis. Exp. (2022)
This video demonstrates an efficient homogenization technique to generate an antigen-adjuvant emulsion to induce experimental autoimmune encephalomyelitis (EAE) in a murine model. The emulsion contains an autoantigen — myelin oligodendrocyte glycoprotein (MOG) — in the water phase, surrounded by a continuous oil phase comprising an inactivated pathogenic bacteria. The emulsion ensures a sustained release of antigens for a prolonged immune response.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
NOTE: A schematic flow of the method is described in Figure 1.
1. Material preparation
NOTE: Prepare all the reagents aseptically in a sterile hood, and aliquot and store at the indicated temperature. The reagents can be stored for up to 2 years without losing their effect.
2. Preparation of CFA/peptide emulsions
3. Quality control of emulsion
Figure 1: Schematic of the process of emulsion preparation using a commercial emulsion kit. This figure has been reused from Topping et al with permission.
Figure 2: Characterization of emulsions produced using different methods. (A) Representative images from three different preparation methods: standard homogenization method, traditional syringe method, and vortex method. A small amount of emulsion was placed on a glass slide and observed under a phase contrast microscope (400x). Scale bar = 50 µm. A representative preparation is shown of each method from >15 experiments. The average D (the volume weighted mean) was measured with a particle size analyzer to be 0.7 µm for the shaking homogenization method, 1.4 µm for the syringe method, and 5.4 µm for the vortex method. (B) Stability of the emulsion over time was measured using the particle size analyzer. An emulsion of CFA/PBS was prepared with the shaking homogenizer and the emulsion kit, and the particle size determined, using laser diffraction over time. One sample was analyzed immediately and then the emulsions were stored at 4 °C. These samples were analyzed 1, 2, 4, and 6 weeks after preparation. The average D volume weighted mean for all samples was 0.73 µm ± 0.03 µm (SD). This figure has been reused from Topping et al with permission.
Supplemental File 1: Calculation of the amount of each reagent (MOG35-55 peptide, PBS, CFA, and IFA) for emulsion preparation. Please click here to download this File.
The authors have nothing to disclose.
1 mL Injection syringe | B. Braun | 9166017V | |
1 mL Injection syringe | Sigma-Aldrich | Z683531 | |
7 ml empty tubes with caps | Bertin-Instruments | P000944LYSK0A.0 | 7 mL tube |
50 mL sterile centrifuge tube | Fisher Scientific | 10788561 | 50 mL tube |
Dispersant, light mineral oil | Sigma-Aldrich | M8410 | Store at RT |
Emulsion kit | Bertin-Instruments | D34200.10 ea | Containing a tube, cap, and plunger |
Incomplete Freund's Adjuvant | Sigma-Aldrich | F5506 | Store at +4 °C |
Mycobacterium tuberculosis, H37RA | Fisher Scientific | DF3114-33-8 | Store at +4 °C |
Minilys-Personal homogenizer | Bertin-Instruments | P000673-MLYS0-A | Shaking homogenizer |
MOG 35-55 Peptide | Innovagen | N/A | |
Montanide ISA 51 VG | Seppic | 36362Z | FDA-approved oil adjuvant |
Pall Acrodisc Syringe Filters 0.2 μm | Fisher Scientific | 17124381 | Sterile filter |
PBS, Ca2+/Mg2+ free | Thermo Fisher Scientific | 14190144 | PBS |
Phase-Contrast Microscope | Olympus | BX40-B | |
Steel Beads 3.2 mm | Fisher Scientific | NC0445832 | Autoclave and store at RT |