Name: a rapid, simple, and standardized homogenization method to prepare antigen/adjuvant emulsions for inducing experimental autoimmune encephalomyelitis
Uploaded: 2022-12-09T14:00:00
Description: Watch this Scientific Journal Video about A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis at JoVE.c

The author would like to acknowledge the animal housing units at Lund University, Camilla Bj&#246;rkl&#246;v and Agnieszka Czopek, for their support, and Richard Williams, Kennedy Institute of Rheumatology, University of Oxford, UK, for constructive criticism and linguistic support producing this manuscript.

All animal procedures were performed according to the practices of the Swedish Board of Animal Research and were approved by the Animal Ethics Committee, Lund-Malm&#246;, Sweden (Permit number: M126-16).
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 tempe...

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Water-in-oil emulsions, such as antigen/Freund&#39;s adjuvant, have been used for more than half a century to induce EAE17. There is currently no standardized method to prepare antigen emulsions that is independent of human influence. Manual mixing using syringes is standard for most laboratories, however this method is time consuming, often results in an excessive loss of material, and the quality differs depending on the scientist preparing it.
The method presented in...

A breakdown of immunological tolerance can result in the generation of autoimmune disorders, such as multiple sclerosis (MS). It is estimated that 2.8 million people are living with MS worldwide1. Although the exact cause of MS is still largely unknown, dysregulation of autoreactive T and B cells, as well as defects in Treg function, play important roles in the pathogenesis of the disease2,3.
Animal models of autoimmune diseases are essential tools to investigate potential therapeutic modalities. The experimental autoimmune encephalomyelitis (EAE) model has been used for almost a century by researchers interested in MS4. In early experiments, the incidence of the disease was relatively low. The introduction of complete Freund&#39;s adjuvant (CFA), containing Mycobacterium and pertussis toxin, enabled the consistent induction of EAE in mice4. Most importantly, it is necessary to mix CFA with a central nervous system (CNS)-specific antigen to generate a homogenous water-in-oil emulsion for inducing EAE. The most common currently available EAE models are based on the active immunization of mice with encephalitogenic peptides. The genetic background of the mice plays an important role in disease susceptibility, with myelin oligodendrocyte glycoprotein (MOG35-55) and myelin proteolipid protein (PLP139-151) peptides used to induce EAE in C57BL/6J and SJL mice, respectively5. However, other mouse strains and CNS-derived peptides can also be used.
The quality of the CFA/peptide emulsion is a critical factor that determines disease penetrance in the active immunization EAE model6. A homogeneous water-in-oil emulsion must be prepared by mixing the encephalitogenic peptides dissolved in aqueous buffer with CFA, otherwise animals will not develop the disease. Numerous protocols have been published on the preparation of CFA/peptide emulsions. Examples include the use of a vortex7, sonication8, syringes and a three-way T connector9, or one syringe only5. However, all these methods are difficult to standardize and are often associated with lengthy and complicated protocols.
Compared to all the above methods, the simple method described here for emulsion preparation offers the advantages of having no person-to-person differences and being relatively fast. The emulsion is generated by a homogenizer shaking the reagents with a set speed, time, and temperature, ensuring fast and consistent results. In addition to inducing disease in the EAE model, this method can also be used to study other autoimmune disease models such as collagen-induced arthritis (CIA) and antigen induced arthritis (AIA)6. Therefore, it is anticipated that this method can be used to consistently induce disease in other animal models that depend on water-in-oil emulsions with autoantigens, such as experimental autoimmune neuritis (EAN)10, experimental autoimmune thyroiditis (EAT)11, autoimmune uveitis (EAU)12, and myasthenia gravis (MG)13. This method also induces general immune responses such as delayed-type hypersensitivity (DTH) consistently6, and could therefore be used for delivering cancer and malaria vaccines (see discussion).
Thus, a rapid (total preparation time ~30 min), simple (all reagents can be prepared in advance and stored), and standardized (the emulsion is accomplished using a shaking homogenizer) method has been developed and is presented here. The CFA/antigen emulsions prepared using this protocol consistently induce disease in autoimmune animal models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Injection syringe</td>
			<td>B. Braun</td>
			<td>9166017V&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Injection syringe</td>
			<td>Sigma-Aldrich</td>
			<td>Z683531</td>
			<td></td>
		</tr>
		<tr>
			<td>7 ml empty tubes with caps</td>
			<td>Bertin-Instruments</td>
			<td>P000944LYSK0A.0</td>
			<td>7 mL tube</td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tube&#160;</td>
			<td>Fisher Scientific</td>
			<td>10788561</td>
			<td>50 mL tube</td>
		</tr>
		<tr>
			<td>Bordetella pertussis toxin</td>
			<td>Sigma-Aldrich</td>
			<td>P2980</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dispersant, light mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Emulsion kit</td>
			<td>Bertin-Instruments</td>
			<td>D34200.10 ea</td>
			<td>Containing a tube, cap, and plunger</td>
		</tr>
		<tr>
			<td>Incomplete Freund&#39;s Adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;F5506</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis, H37RA</td>
			<td>Fisher Scientific</td>
			<td>DF3114-33-8</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Mastersizer 2000&#160;</td>
			<td>Malvern Panalytical</td>
			<td>N/A</td>
			<td>Particle size analyzer</td>
		</tr>
		<tr>
			<td>Minilys-Personal homogenizer</td>
			<td>Bertin-Instruments</td>
			<td>P000673-MLYS0-A</td>
			<td>Shaking homogenizer</td>
		</tr>
		<tr>
			<td>MOG 35-55 Peptide&#160;&#160;&#160;</td>
			<td>Innovagen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Montanide ISA 51 VG</td>
			<td>Seppic</td>
			<td>36362Z</td>
			<td>FDA-approved oil adjuvant</td>
		</tr>
		<tr>
			<td>Pall Acrodisc Syringe Filters 0.2 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>17124381</td>
			<td>Sterlie filter</td>
		</tr>
		<tr>
			<td>PBS, Ca2+/Mg2+ free</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Phase-Constrast Microscope</td>
			<td>Olympus</td>
			<td>BX40-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Beads 3.2 mm</td>
			<td>Fisher Scientific</td>
			<td>NC0445832</td>
			<td>Autoclave and store at RT</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>648463</td>
			<td>Store at RT</td>
		</tr>
	</tbody>
</table>

a rapid, simple, and standardized homogenization method to prepare antigen/adjuvant emulsions for inducing experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

The rapid, simple, and standardized protocol for the preparation of CFA/MOG emulsions is depicted in Figure 1. This method has recently been described elsewhere6. The CFA/MOG emulsions can also be prepared with other methods, such as the traditional syringe method or by vortexing. These methods were compared here by assessing the quality of the emulsions. All the methods produced water-in-oil emulsions; the homogeneity and quality of these emulsions were assessed by a...

Watch this Scientific Journal Video about A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis at JoVE.c

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

Experimental autoimmune encephalomyelitis or EAE is an animal model used to develop new and improved treatments for multiple sclerosis patient. Inducing disease in EAE requires a perfect oil and water emulsion to achieve consistent results. The method of emulsion preparation presented here is free from human influence, thereby limiting inconsistencies from one experiment to another.Even though the method has been optimized for EAE, it can also be used for other autoimmune disease model and also to test cancer vaccines. To prepare the CFA slurry at 100 milligrams of freeze-dried M tuberculosis H37 RA and five, 3.2 millimeters steel beads to a seven milliliter tube. Shake the tube in the homogenizer for 60 seconds at the highest speed setting.Add five milliliters of IFA adjuvant to the tube and shake again for 60 seconds at the highest speed. Then transfer the slurry to a fresh tube with a pipette leaving the beads behind and store at four degrees Celsius until use. To prepare a CFA/Peptide emulsion, download supplemental file one to calculate the amount needed for each reagent.Then place the screw capped tube and reagents to be used on ice. Next, add the emulsion components. First PBS, then the peptide, the CFA slurry.And finally, IFA. Close the tube with the cap firmly by tightening and loosening it several times. Shake the tube vigorously for 5 to 10 seconds by hand to premix the reagents.Place the tube in the shaking homogenizer and secure it with the rod. Set the speed to the highest setting and the time to 60 seconds. Once the run is finished, place the tube on ice for three minutes and repeat the run one or two more times with the same settings.Centrifuge the tube at 300 G from one minute to remove trapped air and compact the emulsion. Remove the cap from the tube. Insert the plunger into the tube, and push it down slowly until it reaches the top of the emulsion.Remove the snap off enclosure at the bottom of the tube by twisting it. Next, remove the plunger from all injection syringes and add a needle to the injection syringes. Then attach the back of the injection syringe to the dedicated lock at the bottom of the tube and lock it with a short twist.Transfer the emulsion from the tube to the injection syringe by pushing the plunger gently. Stop when the emulsion reaches the 0.15 milliliter graduation of the injection syringe. Separate the injection syringe from the tube and insert the plunger carefully taking care that no air enters this syringe.Push the plunger until the emulsion comes out of the needle and repeat the process for the rest of the emulsion present in the tube. To analyze the size of the water and oil particles add a tiny drop of the emulsion to a microscope slide, smear it out with a cover slip, and then push hard in a circular motion to flatten out the emulsion. Examine the smeared emulsion under a phase contrast microscope with 400 times magnification and focus on a field with a monolayer of the emulsion.Ensure that small, uniform, gray or white particles are visible. Emulsions produced by the homogenization method displayed gray or white bean shaped particles representing tiny droplets of water and oil emulsions. In contrast, the emulsion droplets produced using the syringe method were larger and wider.In the vortex method, large gray or black particles were observed corresponding to air bubbles trapped in the emulsion. Changes in particle size distribution over time measured using a laser defraction particle size analyzer for water and oil emulsions did not show any change in particle size over a period of six weeks. The fresh 3 week old and 15 week old emulsions all induced similar EAE diseases in all animals tested throughout the experiment.Mice developed a similar disease pattern in all experiments revealing that the homogenization method presented here produced emulsions that induced EAE in a consistent and reproducible manner. Before making the emulsion for an experiment, download supplemental file number one to calculate the amount of each component needed to minimize waste. The method presented here could be used to standardize emulsion preparation not only for preclinical research, but also in the future to prepare cancer vaccines in the clinic.

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Induction of Experimental Autoimmune Hypophysitis in SJL Mice

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Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

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Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

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Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

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