Contact a company called Hooke Laboratories. They make the adjuvant with the MOG peptide and depending on the incidence and severity of disease in your particular mice they will make stronger does of the peptide in adjuvant already made up.

And one more thing,

I am suspecting my MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) might be the problem. I customized this peptide with MOG35-55 sequence from one of the peptide synthesizing companies so there is no proof that this product will certainly induce EAE, although the company sent me the HPLC result. Is there a way to check this synthesized MOG35-55 is suitable for EAE induction in vitro?

I attached my PhD thesis. You can find in methods section that I had a bit different protocol. It worked very nicely.

Peptide should be no problem if you trust the company.

One might think about SPF2. Afaik: mice that are 'too sterile' are resistent to EAE induction.

Further more: 12 wks old never worked for me.

Were mice moved short before induction? Give them one week to relax. If they are too stressed induction won't work as well.

Britta

Contact a company called Hooke Laboratories. They make the adjuvant with the MOG peptide and depending on the incidence and severity of disease in your particular mice they will make stronger does of the peptide in adjuvant already made up.

But be aware. I don't remember the exact paper, but they said that different amounts of MOG lead to different 'types of eae' as one was then rather Th1 driven, the other Th17

...keep an open mind :-)

Another possibility is the immunization condition might be too strong, which quickly induced Treg cells that suppressed the EAE development (A parameter of bodyweight loss might indicate this).  To clear this, one can do a titration of pMOG, using 50, 100, 150, 200, 300 ug.  From my experience, a low dose of 50 ug can induce EAE in B6 mice.    

The protocol looked fine to me however one possibility is dose of MOG peptide as we generally have 100ug of peptide per 200ul of emulsion. It's not clear what a out of peptide and HKMT you had in 200ul of emulsion. Although there is a variability in disease incidence in different environment, I will repeat experiment once more. Doses of pertussis toxin have major impact of disease severity. So you can play with dose of that and might increase it.If you are concerned about peptide, sacrifice these immunized mice and check for T cell recall response against MOG peptide. So before panicking just repeat the experiment in few mice and may be just change doses of pertussis toxin. Good luck and email me if you have further questions. Good luck

As Sandra says you could try getting pre-made emulsion from Hooke labs. Another thing is make sure your emulsion is REALLY thick (we thicken by pumping up and down in a 1ml syringe and place a drop on water to check it doesn't disperse) before you inject sub-cut if it isn't the immunogen/emusion will dissipate and you won't get a response. In my experience this simple step is where most people go wrong.

I agree with Sandra, the Hooke kit works perfect.

Using younger female BL6 mice (4-6 wks) for inducing EAE would be helpful. 

I successfully induced thousands of mice in the nineties at KU Leuven, Belgium (PhD thesis). At that time, I used mostly mouse spinal cord homogenate MSCH, (see my papers from J Neuroimmunol and Transplantation) but also proteolipid protein (PLP, see paper in Bone). I was using a homogenate with complete Freund's adjuvant enriched with mycobacterium. I was injecting the emulsion of CFA with lyophylized mouse spinal cord homogenate or PLP with a tiny syringe in the hind footpads bilaterally, without anaesthesia. I was immobilizing the animal footpad by introducing it in a slot made in a plastic trasnparent vertical rectangle on a metal support, so the mice could see me,but not reach me :-) .I was working with a technician that was immediately injecting iv the pertussigen using a similar rectangle, by introducing the tail in the rectangle's slot, heating it with infrared light until blood vessel dilation and then injecting iv the aqueous solution. No anaesthesia again. on day 2 we repeated the pertussigen injection in the tail vein. We had experiments of upto 120 mice each, without any problems. All mice develpoed severe, clinically quantifiable paralysis, also histologically noticeable in the brain. MSCH conferred a more severe disease, mostly lethal, paralysis was starting on day 10 after homogenate injection in the footpads. We used SJL mice. Function of the quantity of bordetella pertussis and mycobacterium we could induce severe, lethal disease, or could reinduce the disease after spontaneous cure in a chronic relapsing model (see Acta Endocrinologica). In conclusion - carefully choose a disease sensitive type of mouse, use complete Freund's adjuvant enriched with mycobacterium and a stronger encephallitogenic antigen, inject the antigen emulsion in the hind footpads and the bordetella pertussis iv in the tail veins, trey a simple way to immobilise the footpads and tails, in order to by pass anaesthesia and it will certainly work! Good luck, if problems please contact me at [email protected] !

Everything looks good based on your protocol but the quality of MOG peptide and Pertussis are very important factors for EAE . I used 200 µg MOG35-55 (Gen Script, Piscataway, NJ, USA) and 200 ng pertussis toxin (Sigma Aldrich, St. Louis, MO, USA). Good luck and let me know if you have more questions.

I successfully induced thousands of mice in the nineties at KU Leuven, Belgium (PhD thesis). At that time, I used mostly mouse spinal cord homogenate MSCH, (see my papers from J Neuroimmunol and Transplantation) but also proteolipid protein (PLP, see paper in Bone). I was using a homogenate with complete Freund's adjuvant enriched with mycobacterium. I was injecting the emulsion of CFA with lyophylized mouse spinal cord homogenate or PLP with a tiny syringe in the hind footpads bilaterally, without anaesthesia. I was immobilizing the animal footpad by introducing it in a slot made in a plastic trasnparent vertical rectangle on a metal support, so the mice could see me,but not reach me :-) .I was working with a technician that was immediately injecting iv the pertussigen using a similar rectangle, by introducing the tail in the rectangle's slot, heating it with infrared light until blood vessel dilation and then injecting iv the aqueous solution. No anaesthesia again. on day 2 we repeated the pertussigen injection in the tail vein. We had experiments of upto 120 mice each, without any problems. All mice develpoed severe, clinically quantifiable paralysis, also histologically noticeable in the brain. MSCH conferred a more severe disease, mostly lethal, paralysis was starting on day 10 after homogenate injection in the footpads. We used SJL mice. Function of the quantity of bordetella pertussis and mycobacterium we could induce severe, lethal disease, or could reinduce the disease after spontaneous cure in a chronic relapsing model (see Acta Endocrinologica). In conclusion - carefully choose a disease sensitive type of mouse, use complete Freund's adjuvant enriched with mycobacterium and a stronger encephallitogenic antigen, inject the antigen emulsion in the hind footpads and the bordetella pertussis iv in the tail veins, try a simple way to immobilise the footpads and tails, in order to by pass anaesthesia and it will certainly work! Good luck, if problems please contact me at [email protected] !

I have a problem with inducing EAE in mouse. What could I do? - ResearchGate. Available from: https://www.researchgate.net/post/I_have_a_problem_with_inducing_EAE_in_mouse_What_could_I_do#56716b96614325cea98b4567 [accessed Dec 16, 2015].

I agree with Gareth, the thickness of the emulsion is very important, once injected it should stay (you'll see a white bubble or ball) in the place of injection, it shouldn't disperse (otherwise it will not work). To help seeing that you can clean the skin with alcohol, and move the hair to the sides. In order for the injection to be really subcutaneous, the needle should be parallel to the skin while going in (if it's too deep it won't work again). Also, since you're injecting the flanks, take care to not to inject where the inguinal ganglia are (it should be a little upper). I also agree with other comments about the amount of MOG, you should try different concentrations. Also the toxin is important, be sure it's not too old. Finally, don't give up, try a couple more times. To learn how to induce EAE (and to make it consistent) takes time, you'll see the incidence of EAE in your animals will increase with time. To be sure you're activating the right type of immune response you can check if cells from the inguinal ganglia proliferates in the presence of MOG and, if you can, you can do some general staining (like H&E) in the spinal cord to see if there are infiltrates/inflammation/demyelination. Please feel free to contact me if you have further questions. Good luck!!

You could try increasing the pertussis toxin doses. I get 90% incidence with 400 ng / 200 ul doses on day 0 and day 2. The activity of pertussis will vary from lot and company and this is something you will need to determine empirically. 

Hi,

It may seem an unnecessary question, but are sure of your mice background? Are they for sure pure B6 expressing H-2b?

Dumitru Branisteanu wrote,

"In conclusion - carefully choose a disease sensitive type of mouse, use complete Freund's adjuvant enriched with mycobacterium and a stronger encephallitogenic antigen, inject the antigen emulsion in the hind footpads and the bordetella pertussis iv in the tail veins, try a simple way to immobilise the footpads and tails, in order to by pass anaesthesia and it will certainly work!"

Certainly in the UK, we are not allowed to immunise in the footpads any more (and perhaps elsewhere?) on ethical/animal welfare grounds. Inject into the flank sub-cut and it works fine. Also pertussis injection should be intraperitoneal, intravenous is unnecessary in my experience.

Before hassling with the MOG preparation (which sounds like you're doing it fine), try injecting the pertussis by tail vein on each day (200ng in 100µL per injection). We had dramatic improvement in disease induction by injecting tail vein rather than i.p. Also, you can do MOG injection, immediately followed by pertussis. You don't have to wait 2 hours. And, you should be able to do all of these injections without anesthetizing.

FYI, MOG-induced EAE also develops in the absence of PT...

Buy the EAE induction kit from Hooke Laboratories as it works like a charm.

Why is it more ethical to inject in the flanks than in the footpads? This is a dilemma mice themselves wouldn't understand.....

Most of the time, PT is the problem. Change the manufacturer. Minor point: there is no need to wait 2 to 5 h to inject PT after immunization.

Thank you all!!

I listed up all the main points that you guys suggested. (Modify MOG, PTX dose, mouse age; use younger mouse, injection method.. etc…)

I will re-design the experiment according to your suggestion and repeat it.

Hope my next posting will contain good news!

p.s I used C57b/6NCrl jori from Charles River. The company indicates that the MHC haplotype for this strain is H2b.

Methods

Commercial Freund's complete adjuvant (Sigma, USA)

- 55-35 MOG peptide sequence (MEVGWYRSPFSRVVHLYRNGK) (Alexis, USA)

Pertussis toxin (Sigma, USA)

PBS buffer

Diethyl ether to stun the animal ((Merck, Germany

Since the concentration of complete Freund's adjuvant Commercial tuberculosis in 1mg / ml and to induce EAE is low, it should at least tuberculosis concentration and concentration 4mg / ml scored.

Freund's complete adjuvant at least twice in the Eppendorf at high speed (12,000 around 12 minutes) were centrifuged and the supernatant was removed after each centrifuge half and replace it with a new adjuvant was added and the volume. After the last centrifugation step, half of the supernatant was removed and the lower half thick was used as an adjuvant.

250 µg MOG on volume of 100 µl PBS with adjuvant volume concentration (numbers per mouse) interface between two syringes 2 ml taken consecutively until well blended. To achieve this objective the displacement of at least one hour a mixture between two syringes should be performed. Then the combination of insulin by syringe subcutaneously in the flank region of mice pre-treated cotton by anesthesia is injected. Immediately after the injection, as well as 48 hours later, 500 ng pertussis toxin in the volume of 200 µl PBS was injected intraperitoneally (Tsvyr2-3) and then daily until day 20 after the induction of clinical symptoms and weight control mice Submitted. Usually the symptoms of EAE, 12 to 14 days after injection appears MOG.

be successful and victorious

Hello, if these protocols could be helpful to you,  I will be glad of your success.

Do not hesitate to contact me if you will need more advice.

To Dumitru Branisteanu

Well mice don't walk on their flanks for a start and I'm sure the mice appreciate not having to walk on swollen inflamed hind footpads.  I'm not sure what EU policy is on this but it is banned in the UK and in a number of other countries.

Regarding the use of EAE, you should take into account the enclosed. There are also other models that might be better used mentioned in the report. Search the authors for their latest modelling of MS, especially Daniel Anthony (Oxford).

J Pharmacol Toxicol Methods. 2013 May-Jun;67(3):169-76. doi: 10.1016/j.vascn.2013.01.009. Epub 2013 Jan 26.

Reducing suffering in experimental autoimmune encephalomyelitis (EAE)

See EAE protocol that I have used, step by step.  Good luck!!!

Material: Bordetella pertussis toxin (Sigma-Aldrich, St Louis): Prepare stock solution  50 mg/ml in PBS sterile inside hood. Remember we will inject Pertussis toxin i.p., 300 ng/200ml, twice/animal, on the day of immunization (just after MOG injection) and 48 hours apart.

MOG: (the amount injected depends how pure is the protein). We used MOG35-55  150mg/100 ml/animal, but for each new lot that you receive you have to check what is the best amount.

CFA + M. tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA): 4mg/ml

Mice C57Bl/6 females, age 8-12 weeks, weight 20 g (relevant animals reach minimum 20 g).

Methods:

1) MOG should be weigh (in small plastic dish) outside the hood, but after we have to dilute in PBS inside hood. Use sterile tip for automatic pipette. Use gloves and mask to avoid inhalation.

2) Pertussis toxin should be dilutes insides de hood as well.

3) For the mix CFA + M. tuberculosis, we use the mortar to grind the M. tuberculosis before weigh and dilute in CFA.

4) After diluted the MOG in PBS, transfer the volume to eppendorf tube. After, transfer from the eppendorf tube to a GLASS syringe (3ml) sterile using 18G ½ needle. This syringe should be connecting to a stopcock classic ( three ways). Push the volume inside the syringe until the connection of the stopcock. Measure what is the volume that you got it.  Mix very well CFA + M. tuberculosis. Now, fill up a second 3 ml GLASS syringe with the solution CFA + M. tuberculosis, same volume that you got previously for MOG.

5) Now close third exit of the stopcock, and mix both volumes very well, the MOG and the CFA + M. tuberculosis. Repeat the same procedure over 15 minutes, with intervals of 3 minutes keeping on ice. After 15 minutes, to verify if the emulsion is ready for injection, do the follow test:  place a drop of the emulsion on water and check that if the emulsion remains condensed as a drop or spreads. The ideal consistency is to remain as a drop.

6) Once we have the emulsion and Pertussis toxin preparedl, we go to clean area. We take the solutions and 1.0 ml syringes and 26G 3/8 needles for Pertussis toxin, i.p. injections, and 25G5/8  needles for subcutaneous injections (in the tail base) of emulsion (CFA + M. tuberculosis + MOG). Remember after 48 hours repeat Pertussis toxin injection.

7) Animal weight and clinical score were monitored daily as previously described (dos Santos et al., 2005, 2008; Bernardes et al., 2013, 2015). The scores were defined as follows: 0 = no clinical signs, 1 = tail paralysis (or loss of tail tone), 2 = tail paralysis and hind-limb weakness, 3 =hind-limb paralysis and 4 = complete hind-limb paralysis and frontlimbweakness. This protocol promoted the development of EAE clinical signs, which were initially observed at 9–10 dpi and peaked at 13–14 dpi.

To Gareth Pryce

Ethical issues may have evolved since my last paper from 2007 dealing with the model (J Steroid Biochem Mol Biol) and people from the lab of

KU Leuven may not be as human as British researchers. However, mice were running just fine after the injection in the footpads. I couldn't say the same after 10 days, when they were getting paralysis and crawling on their belly or flanks....

Flippancy is never a good look Dumitru. I've seen both of arthritis and ulceration after footpad injection. We tend to take things rather more seriously in the UK.

Indeed, neither is posing as a better Christian than the Pope. I never saw the above mentioned issues in the over thousand mice I worked with. Maybe it is also a matter of injection skills. Do you see MS patients, Gareth?

Footpad injections will confound your clinical scores. Inject subcutaneously at the base of the tail as folks here have previously suggested. 

Jordan Warford

Absolutely no problems in evaluating disease severity with a clinical score after footpad injection in my experience. Motility is not impeded at all after footpad injection with upto 50 microliter emulsion, Moreover, disease incidence is close to 100% when emulsion of encephalitogenic trigger in CFA enriched with mycobacterium tuberculosis in the footpads and pertussigen tail iv injections are used on days 0 and 2 (Dale McFarlin protocol), The effectiveness of induction is easy to see (there is no hair on the footpads!) and protocol ease allows using more animals in one experiment. I accept, however, the ethical arguments of  Michael Seed and Gareth Pryce, and if induction can be done somehow less harmfully but as efficient, then.... why not? Ethical issues stop, however, in ten days, when severe paralysis occurs. Animals should be then helped with access to food and water.

Kylie Yong

Here is a proposal of a simple clinical score of severity for paralysis assessment, I hope it would be of some help for you once you start inducing the disease

0 - no signs or symptoms

0.5 - partial limp tail or trembling, lack of coordination, mild weakness, eye inflammation

1 - limp tail, no difficulties in recovery to upright position after tail twisting

2 - limp tail, mice can still walk, but difficulties in recovery to upright position after tail twisting

3 - limp tail and severe impairment of mobility or paralysis of hind feet, mouse cannot walk, but can still crawl

4- total paralysis or global severely damaged mobility and bad general condition, with impossibility to crawl, or death after clinical signs of paralysis

Please take note for everyone inducing EAE using footpad injections....as some of you mentioned above, this technique is not allowed....you will not get your papers published if you use this route!

As Sandra says footpad injection is not only no longer allowed, it is completely unneccessary. 

I am sorry for having interfered regarding this item, I was just trying to help. Things may indeed have changed since my last papers in Bone 2003 and J Steroid Mol Biol in 2007 when I induced EAE witn completely unnecessary footpad injections at KU Leuven. Good luck with your experiments, everybody!

I might be repeating what others had said but, still I know how frustrating it is not to succeed with the EAE induction, so, I want to share my own version. As Nemat said age is very important. In my experience 8 to 12 weeks old is good, but this range could be strain dependent. I am more successful getting all my mice sick and relatively synchronous when I use 10 week old mice. After 12 weeks you start dealing with issue of immune senescence that its also strain and sex dependent. 

As Sandra mentioned the amount of MOG should be titrated specially when you are housing your mice in SPF conditions. Every time I moved to different vivaria I need to do a myelin peptide titration no matter if I am dealing with B6, SJL (using PLP) or hybrids susceptible to either MOG or MBP. In my case a 1:10 ratio of MOG: HKMTb works great (ex. 50 ug MOG: 500 ug HKMTb in 1:1 volume). I never used pre-made CFA, I like to grind my HKMTb in IFA, mine are from Gibco.

I also prefer to inject in 4 sites: flanks and shoulderblades. I like to spray the mouse with 70% ethanol and wipe the fur against the hair growth. (Some like to shave the mice but that causes more irritation, I don't do it). As mentioned before, be careful about how you introduce the needle making sure your emulsion is in the subcutaneous compartment and then wipe the fur to be sure there is no emulsion on it. If so the mouse will lick the emulsion and will develop tolerance to the myelin peptide.

I haven't used Hooke kit, so, I can give you any advice about it. Regardless, peptide quality is critical. I prefer Genscript but, I always ask >98% purity and to aliquot the "bulk" order in tubes or vials of 10mg each. That way the peptide does not suffer with freezing-thawing-freezing. Additionally, I prefer to dissolve the peptide in 1X PBS, sterile. As stated by many, the emulsion needs to be "toothpaste" stiff and ensure that when you load the syringes there are no air bubbles. I know many people that prepare a very stiff emulsion and failed in this part, meaning they were not injecting the right amount and mice were not getting sick. You need to develop the "eye" of looking at the syringe against a lightbulb to see the translucent bubble versus the white emulsion. 

Lastly, the pertussis has to be good quality and relatively fresh. Shelf life of PTX is short. I use List Biologicals and prepare a "stock" of 50ug/ml in sterile injectable saline. Then, I prepare a fresh working solution every time I am injecting mice. I keep the "stock" for only 3 months. In my experience, after 3 months its bioactivity is compromised. As with the emulsion bubbles I know many incidents of failed EAE induction caused by an "old" pertussis.

Good luck in you next induction!

Lillian

Hi

My protocol is as follows:

Freund adjuvant per mouse was mixed with 100 ml double the volume removed and centrifuged for ten minutes away ten thousand, and then supernatant half And again with 100 ml adjuvant added to it and this operation was repeated four times to Santryfyvzhra high concentrations of mycobacteria be added at the end of the supernatant and remains only 100 ml.

MOG35-55 mixed with 100 ml sterile PBS And then two syringes connected with the photos I sent it to you adjuvant And peptide for one hour slowly and with caution mixture And then injected into the body sides and the pertussis toxin immediately injected intraperitoneally your way out.

The 10 day after inducing EAE the tail subjective symptoms such as paralysis appear, but the main condition is that After inducing EAE, another person close to the animals and do not stress your need to water them and feed only.

Kylie, have you or anyone at you place ever had a positive induction of EAE? If not, the housing conditions of your mice might be too "clean". If that is a possibility, try to get fecal material from cages in institutes, where mice with EAE live. The mouse intestinal microbiome is decisive to allow induction of EAE. 

Check out our papers for induction of EAE and in particularA l-Izki S, Pryce G, O apos Neill JK et al. (2012) . Practical guide to the induction of relapsing progressive experimental autoimmune encephalomyelitis in the Biozzi ABH mouse.Multiple Sclerosis and Related Disorders vol. 1, (1) 29-38.

10.1016/j.msard.2011.09.001.  Good luck

Hi Yong. 

The quality of emulsion is critical. Complete Freund Adjuvant is used in emulsion for at least 2 reasons.

No. 1, boosting immunological response. No. 2, working as an slow release formulation.

I agree Gareth that thickness of emulsion is critical and we should check the thickness of emulsion by some objective indicators.

Emulsion is a stabilized mixture (dispersion) of two or more immiscible liquids, like oil and water, and emulsion is widely used in food and cosmetic industry.  For standardizing and increasing the stability of emulsion, high pressure and high temperature are usually applied to increase the stability of emulsion. Besides this, chromatography is used for checking the diameter of emulsion particles.

One method is good for checking the quality of emulsion: Place the MOG-CFA emulsion at 4 degree overnight. If you find the water and oil do not separate and keep the same status as the day before, the emulsion should be good enough for EAE induction.

Good luck.

 LI

Hi, I know this is an older thread, but I found it because I have recently been having problems with my EAE (have been doing the MOG model successfully for 3 years and have never had a problem before). It seems that the consensus is that the PT could be the culprit, and I have been using my current batch for ~6 months. However, I have done this before and have never had absolutely no mice develop as I experienced this time (out of 12 mice!)

We use the 50 ug lyophilized PT from List Biologicals (Product #181). I make a 2.5 ug/mL stock in sterile saline and dose the mice on Day 0 and 2 with 500 ng of PT.

My question is -- does anyone use a different PT? Or possibly store it differently? It seems like I will end up wasting some of the solution each time if I can only use it for 3 months at a time and from List I can only order 50 ug at a time, not a smaller vial.

Thanks in advance for any advice!

Dear Kylie,

Assuming that you might still be interested in this somewhat aged thread I want to add a short comment.

A suprisingly high percentage of people working with C57Bl/6 mice are not aware that there are two major substrains, B6-J and B6-N, which have been shown to differ in a variety of genetic parameters and also show different outcomes in several experimental settings. We ourselfs could identify differences in their responses in some immunological models, where - upon analysis of knockout mice with an non-matching background - this may lead to misinterpretation of results.

I seem to remember that some time ago former colleagues of ours had problems in getting their EAE experiments running if they used the "wrong" C57Bl/6 substrain. Might be worth checking out which strain your department is purchasing and which strain is used by colleagues who's EAE works fine. (If they actually know).

Best regards,

Markus