Fight Aging!

This lengthy post covers the topic of setting up and running a self-experiment, a human trial of a single individual, to assess whether a ten week course of flagellin immunization will significantly and beneficially affect gut microbe populations. Flagellin is the protein making up a flagellum, the appendage that bacteria use to move themselves around. As it happens, the presence of flagellae correlates decently well with harmful gut microbes, and the absence of flagellae correlates decently well with helpful gut microbes. In principle, provoking the immune system into greater efforts to chase down and destroy anything with a flagellum will better manage the microbial populations of the gut. These populations change with age in ways that promote chronic inflammation and reduce the generation of beneficial metabolites.

Flagellin has been used in human clinical trials as an adjuvant to improve vaccination efficacy: it is recognized by the immune system, and helps produce a greater immune response. It was shown to have only minimal side effects in those trials. There is no human data for effects on the gut microbiome, but a very interesting paper describes the effects in mice. The outcome is a shifting of the relative population sizes of gut microbes in a beneficial direction that will reduce chronic inflammation. It is unknown as to how long such an effect might last - whether rousing the immune system is a short-term process, or whether it will continue to better guard against unwanted microbes.

The purpose in publishing this outline is not to encourage people to immediately set forth to follow it. There are, for example, important caveats in the above mentioned mouse study regarding links between flagellin immunity and human gut diseases. If you come away thinking that you should just jump in, and as soon as possible, then you have failed at reading comprehension. This post is intended to illustrate how to think about self-experimentation in this field: set your constraints; identify likely approaches; do the research to fill in the necessary details; establish a plan of action; perhaps try out some parts of it in advance, such as the measurement portions, as they never quite work as expected; and most importantly identify whether or not the whole plan is worth actually trying, given all that is known of the risks involved. Ultimately that must be a personal choice.

Contents

• Why Self-Experiment with Flagellin Immunization?
• Caveats in More Detail
• Summarizing Flagellin Immunization
• Establishing Dosage
• An Introduction to Injections
• Considering Autoinjectors
• Obtaining a Needle-Free Injection System
• Obtaining Vials of the Correct Size
• Preparing Flagellin for Injection
• Obtaining Flagellin
• Storing Flagellin
• Validating the Purchased Flagellin
• Establishing Tests and Measures
• Guesstimated Costs
• Practice Before Working with Flagellin
• Schedule for the Self-Experiment
• Where to Publish?

Why Self-Experiment with Flagellin Immunization?

Gut microbes are an important influence on long-term health and aging. If forced to guess, they might be in the same ballpark as exercise. The first changes occur around age 35, reducing the generation of beneficial metabolites. Later changes are more detrimental. There is at present little that people can do to reliably influence gut microbe populations, beyond improving diet, there is considerable variability between individuals, and only a few services such as Viome that offer assays to assess progress. Any sort of therapy that works for a majority of people without aggressive customization would be a step forward, and responsible self-experimentation can help to determine whether this is a viable path forward. These reasons must be balanced against a sober assessment of the risk involved in vaccinating oneself with a bacterial protein that has been used in only a few human trials, and an acceptance of personal responsibility for consequences should one choose to run those risks.

Caveats in More Detail

There are two areas of personal responsibility to consider here. Firstly, this involves injecting a bacterial protein that has little published data on human use (even when that data shows good safety in the short term). As the mouse study paper points out, human patients with inflammatory bowel disease exhibit greater immune reactivity to flagellin, and it is unclear as to whether this drives disease pathology or is a beneficial adaptation. That is a leap into the unknown. So is crossing the road, or indeed getting out of bed in the morning, but there are definitely different degrees of risk and comfort.

Secondly, obtaining flagellin in the manner described here is potentially illegal: not yet being a formally registered medical treatment, it falls into a nebulous area of regulatory and prosecutorial discretion as to which of the overly broad rules and laws might apply. In effect it is illegal if one of the representatives of the powers that be chooses to say it is illegal in any specific case, and there are few good guidelines as to how those decisions will be made. The clearest of the murky dividing lines is that it is legal to proteins that are not defined as a therapy for research use, but illegal to market and sell them for personal use in most circumstances. This is very selectively enforced, however, and reputable sellers simply declare that their products are not for personal use, while knowing full well that this is exactly what their customers are doing in many cases.

Choosing to purchase and use flagellin would therefore likely be a matter of civil disobedience, as is the case for anyone obtaining medicines or potential medicines outside the established national system of prescription and regulation. People are rarely prosected for doing so for personal use in the US - consider the legions of those who obtain medicines overseas for reasons of cost, despite the fact that doing so is illegal - but "rarely" is not "never." If you believe that the law is unjust, then by all means stand up against it, but accept that doing so carries the obvious risks of arrest, conviction, loss of livelihood, and all the other ways in which the cogs of modern society crush those who disagree with the powers that be.

Summarizing Flagellin Immunization

Flagellin is classified as a pathogen-associated molecular pattern, something that both innate and adaptive immune systems recognize and react to. The use of flagellin as an adjuvant to spur greater immune response to another, attached protein or protein fragment is an established field of development. This is largely because of the favorable characteristics of flagellin: it doesn't appear to be associated with mechanisms that might cause severe reactions. As noted earlier, human trials have taken place, and the safety data is good - at least over the short time frame in which safety is assessed.

Immunization with flagellin versus its use as an adjuvant is a matter of injecting the protein on its own at an appropriate dose, to rouse the immune system to greater activity against anything connected to flagellin. Given that we mammals, mice and humans, are already sensitized to flagellin, it is interesting that boosting that reaction has a noticable affect on gut microbes. That was only recently demonstrated in mice. A larger literature on this topic has yet to be established.

Establishing Dosage

For small molecule drugs, it is possible to produce a starting point for human dosage given a mouse dose via standard equations, as shown in "A simple practice guide for dose conversion between animals and human". Unfortunately this doesn't apply to vaccines. In fact, it is fair to say that there is no rigorous way to determine how vaccines scale from mice to people, as the systems involved are enormously complex. Outcomes depend on immune system behavior, not on animal size, and modeling the immune system is a speculative activity at best. Many approved vaccines are probably using wildly suboptimal doses.

Insofar as there is any common wisdom, it is that similar doses in mice and humans appear to be a good starting point, based on those cases in which rigorous dose-finding has been carried out. In the case of flagellin, the mouse study employed a 10 μg dose injected weekly for 10 weeks. A 2017 human clinical trial used two monthly doses of 1 to 10 μg via intramuscular injection. Studies in non-human primates used used doses of 1 to 10 μg.

Much larger doses have been given to animals without apparent toxicity, but the failure mode of immunization at too high a dose is intense inflammation or, worse, a cytokine storm that has the potential to be very harmful or even fatal. Whether such adverse consequences are triggered, and how likely they are, is species-dependent. There is no human data for higher doses than those mentioned above, so at the end of the day, the best starting point appears to be to stick with the 10 μg dose repeated weekly over 10 weeks via intramuscular injection, on the basis that this has been trialed in humans.

An Introduction to Injections

The relationship between different forms of injection, dosage, and effects is actually a complicated and surprisingly poorly mapped topic. There are four type of injection to consider, here listed in descending order of difficulty to carry out safely: (a) intraperitoneal, through the stomach muscle into the abdominal body cavity, which is rare in human medicine but common in studies using small animals; (b) intravenous, into a vein, which requires some practice to get right; (c) intramuscular, into the muscle beneath the skin; and (d) subcutanous, into the lower levels of the skin.

The amount of fluid that can be easily injected varies by type. In humans, effectively unlimited amounts of fluid can be introduced via intraperitoneal or intravenous injection. The subcutaneous route is limited to something less than 1 ml, and intramuscular is limited to 2-3 ml depending on location. These are all very fuzzy numbers, but these upper limits don't really matter for the purposes of injecting 10 μg of a protein: it can be dissolved in a very small amount of liquid, 0.5 ml or less.

Different injection routes can alter the character of the injected medicine; how much is required to gain a given effect, how long it takes to get into the system and how fast it does it. A rare few types of medication cannot be injected subcutaneously, because the metabolism of the skin will degrade them, while some are better given subcutaneously. If you root through the literature looking for comparisons between performance and dosage for different injection types, you'll find a very ragged collection of examples showing that there are few coherent rules. Some compounds have no discernible differences between injection route, some see altered peaks of concentration, some require higher doses when subcutaneous, some require lower doses when subcutenous. Oil-based solutions can produce a very slow uptake of medication when injected into muscle or skin in comparison to an intraveous injection, while water-based solutions result in just as rapid an uptake into the bloodstream.

It seems sensible to say that a self-experimenter should try to use the much easier paths of subcutaneous and intramuscular injection, and just keep the same dose as was established for intravenous injection. For most people, intraveous injections require a helper or a lot of painful practice. For subcutaneous and intramuscular injections, there is a market of autoinjection tools that can remove many of the challenges inherent in managing injections. In the case of flagellin, it makes sense to stick with the human trial approach of intramuscular injections.

Considering Autoinjectors

Sticking a needle into one's own flesh is not an easy thing to do, and this is the rationale for the range of autoinjection systems that have been developed by the medical community. They are most easily available for subcutaneous injections; spring-based devices that accept a standard needle and syringe, and that are trigged by a button push. Intramuscular autoinjectors do exist, but unfortunately largely not in a general or easily available way. All of the needle-based intramuscular autoinjectors are regulated devices that come preloaded with a particular medicine, and are not otherwise sold in a more generally useful way. Unfortunately, there is no automation that can help with intravenous injections. You are on your own there.

Option 1: Subcutaneous Autoinjection with Needle and Syringe

If intending to carry out subcutaneous injections it is easy enough to order up a supply of disposible needles and syringes, an autoinjector device that accepts the standard needle and syringe arrangement, and other necessary items such as sterilization equipment from the sizable diabetes-focused marketplace. Such injections are relatively easy to carry out, a wide range of vendors sell the materials, and there is a lot of documentation, including videos, available on how to carry out subcutaneous injections. All of the equipment is cheap. Buying these materials will probably put you on a list in this era of the drug war, but there are many people out there doing it.

Option 2: Subcutaneous or Intramuscular Needle-Free Autoinjection

Are there viable alternatives to needles? As it turns out, yes, and some can solve the problem of missing general intramuscular autoinjectors as well. Needle-free autoinjectors that use a thin, high-pressure fluid jet to punch medication through the skin are a growing area of development. These systems have numerous advantages over needles, but they are more expensive, most can only manage subcutanous injections, and all are limited in the amount of fluid they can inject in comparison to the traditional needle and syringe. Nonetheless, for the purposes of this outline, I'll focus on needle-free systems. The biggest, primary, and most attractive advantage of a needle-free system is in the name: it means not having to deal with needles in any way, shape, or form.

Obtaining a Needle-Free Injection System

There are a fair number of needle-free injectors on the market, but most are hard to obtain unless you happen to be a regulated medical facility running through the standard regulated purchase model, and are looking for large numbers of units in a bulk purchase. Some systems use compressed gas, others use springs. The spring-based systems tend to be less complicated and more reliable. From my survey of the marketplace, the two systems worth looking at are (a) PharmaJet, which can be purchased in the US via intermediary suppliers, and (b) Comfort-in, which is sold directly to consumers in most countries by an Australian group. So far as I can tell, PharmaJet is the only available needle-free system that is capable of intramuscular rather than subcutaneous injection.

PharmaJet is the better engineered and more expensive of these two systems, and its specialized 0.5 ml syringes are built to be one-use only. Further, loading fluid into the syringes requires the use of vials and a vial adaptor. First the vial is loaded with the fluid to be injected, then the vial is connected to the syringe via the adaptor to transfer the fluid. Comfort-in has a similar setup, but is more flexible, and on the whole more consumer-friendly when considering the entire package of injector and accessories. It is has a wider range of vial and other adaptors. Further, the Comfort-in syringes can in principle be reused given sterilization, though of course that is not recommended.

The instructions for both of these systems are extensive, and include videos. They are fairly easy to use. One caveat is that needle-free systems produce a puncture that more readily leaks injected material back out again than is the case for needles. It is a good idea to have a less absorbent plaster ready to apply immediately after injection, such as one of the hydrocolloid dressings now widely available in stores.

Obtaining Vials of the Correct Size

If using the insulin needle and subcutaneous injection approach, then any variety of capped glass vial will do when it comes to mixing and temporarily holding liquids for injection. It does, however help greatly to either use preassembled sterile vials or assemble your own vials with rubber stoppers and crimped caps, as described below, as that sort of setup makes it easier to take up small amounts of a liquid into a syringe. If using the needle-free systems, then vials of a specific type and size are necessary in order to fit the adaptors. The rest of this discussion focuses on that scenario.

There are many, many different types of vial manufactured for various specialized uses in the laboratory. The type needed here is (a) crimp-top vial, also called serum vials by some manufacturers, with (b) a 13mm (for PharmaJet and Comfort-in) or 20mm (for Comfort-in only) diameter open top aluminium cap, one that has a central hole to allow needles and adaptor spikes through, and (c) a rubber or rubber-like stopper that is thin enough in the center to let a needle or adaptor spike past. The cap is crimped on over the rubber seal to keep everything in place - this requires a crimping tool, and removing it requires the use of another tool.

There are two options here. The first option is to purchase preassembled empty sterile vials of the right size and a set of disposable needles and syringes to transfer liquid into the vials. In order to continue to bypass the whole business of needles, however, the other alternative is to purchase vials, stoppers, and aluminium caps separately, or in a kit, and assemble your own vials. A crimping tool is also needed in order to seal the cap. That tool, like the vials and the caps, must be of the right size. Be careful when purchasing online. Vials are categorized by many different dimensions, and descriptions tend to mix and match which dimensions of the vial they are discussing, or omit the important ones. For sterile vials, it is usually only the cap diameter that is mentioned. For crimp-top vials, there are any number of dimensions that might be discussed; the one that needs to match the cap diameter is the outer diameter of the mouth or crimp.

It is usually a good idea to buy a kit where possible, rather than assembling the pieces from different orders, but if taking the assembly path, it is best to buy all the pieces from the same company. Wheaton is a decent manufacturer, and it is usally possible to find much of their equipment for sale via numeous vendors. One can match, say, the crimp-top 3ml vials #223684 with 7mm inner mouth and 13mm outer mouth with snap-on rubber stoppers #224100-080 of the appropriate dimensions and 13mm open top caps #224177-01. Then add a 13mm crimping device #W225302 and pliers #224372 to remove 13mm crimped caps.

Preparing Flagellin for Injection

If using a needle-free injection system, you will likely be limited to injecting 0.5ml amounts. Thus the objective here is to obtain 10 μg of flagellin dissolved in 0.5 ml of phosphate buffered saline in each of ten sealed vials, ready to be used with the injection system, with as little contamination as possible from the environment, and stored a freezer until it is ready to use. Depending on the size of the vial, it might contain doses for multiple injections, but stick to one dose per vial. It is not a good idea to carry out repeated freeze-thaw cycles on protein solutions, which is what you'll have to do if all the doses are in 5ml of solution in one vial. Multiple freeze-thaw cycles degrade the protein.

When ordering flagellin, it will typically arrive in 10 μg to 100 μg vials. Empty 100 μg of flagellin into a single vial holding 5 ml of phosphate buffered saline. Mix well. Place that vial and 10 empty vials into a vial rack. Then use a pipette to transfer 0.5 ml into each empty vial. Seal and crimp each vial as you go. Then put the whole set into the freezer.

Keeping Things Sterile is Very Important

Keeping hands, tools, vials, and surfaces clean and sterile is important: wash everything carefully and wipe down surfaces with an alcohol solution before and after use. Laboratories use autoclaves, which sterilize with steam. These are largely expensive devices, but a range of smaller, cheaper options exists. There are many best practices guides and summaries available online. This extends to the injection itself. Even with needle-free systems, an injection site should still be wiped down with alcohol first. It is all too easy to infect an injection site if skipping the precautions, and this can have severe consequences.

Obtaining Flagellin

Not all flagellin is exactly the same molecule, nor is it created in the same way. The mouse study used a standard option of flagellin protein from Salmonella typhimurium (there are numerous strains, but strain differences are probably irrelevant). This is typically produced using recombinant protein manufacture techniques. There are many established companies that manufacture and sell proteins, including flagellin, to high standards of quality and safety, but as a general rule they will not ship to any customer other than an established and validated lab business. The usual way for everyone else to obtain cost-effective supplies of this sort is to search Alibaba for suppliers who offer that compound in their catalog, but unfortunately this isn't an option for flagellin. Instead one must search Alibaba for protein synthesis companies, pick a smaller one, and negotiate a price for flagellin synthesis.

As noted at the outset of this post, all of these efforts to obtain, ship, and use any random protein for self-experimentation are to some degree illegal - it would be an act of civil disobedience carried out because the laws regarding these matters are unjust, albeit very unevenly enforced. Many people regularly order pharmaceuticals from overseas, with and without prescriptions, for a variety of economic and medical reasons, and all of this is illegal. The usual worst outcome for individual users is intermittent confiscation of goods by customs, though in the US, the FDA is actually responsible for this enforcement rather than the customs authorities. Worse things can and have happened to individuals, however, even though enforcement is usually targeted at bigger fish, those who want to resell sizable amounts of medication on the gray market, or who are trafficking in controlled substances. While the situation with an arbitrary protein isn't the same from a regulatory perspective, there is a fair amount written on the broader topic online, and I encourage reading around the subject.

Open a Business Mailbox

A mailbox capable of receiving signature-required packages from internal shipping concerns such as DHL and Fedex will be needed. Having a business name and address is a good idea. Do not use a residential address.

Use Alibaba to Find Manufacturers

Alibaba is the primary means for non-Chinese-language purchasers to connect to Chinese manufacturers. The company has done a lot of work to incorporate automatic translation, to reduce risk, to garden a competitive bazaar, and to make the reputation of companies visible, but it is by now quite a complicated site to use. It is a culture in and of itself, with its own terms and shorthand. There are a lot of guides to Alibaba out there that certainly help, even if primarily aimed at retailers in search of a manufacturer, but many of the specific details become obsolete quickly. The Alibaba international payment systems in particular are a moving target at all times: this year's names, user interfaces, and restrictions will not be the same as next year's names, user interfaces, and restrictions.

Start by searching Alibaba for protein synthesis companies. There are scores of biotech companies in China for any given specialty. Filter the list for small companies, as larger companies will tend to (a) ignore individual purchasers in search of small amounts of a protein, for all the obvious economic reasons, and (b) in any case require proof of all of the necessary importation licenses and paperwork. Shop around for prices - they may vary widely, and it isn't necessarily the case that very low prices indicate a scam of some sort. Some items and services are genuinely very cheap to obtain via some Chinese sources. Remember to ask the manufacturer for mass spectra and liquid chromatography data if they have it.

Many manufacturers will state that they require a large (often ridiculously large) minimum order; that can be ignored. Only communicate with gold badge, trade assurance suppliers with several years or more of reputation and a decent response rate. Make sure the companies exist outside Alibaba, though for many entirely reliable Chinese businesses there are often sizable differences between storefronts on Alibaba, real world presence, and the names of owners and bank accounts. Use your best judgement; it will become easier with practice.

Arrange Purchase and Shipping via Alibaba

Given the names of a few suppliers, reach out via the Alibaba messaging system and ask for a quote for a given amount of flagellin; you will have to provide the sequence and a reference to the paper in which it is described. Buy more than you'll think you need, and make sure it is packaged into multiple vials, as one vial will be used to validate the identity and quality of the batch. Payment will most likely have to be carried out via a wire transfer, which in Alibaba is called telegraphic transfer (TT). Alibaba offers a series of quite slick internal payment options that can be hooked up to a credit card or bank account, but it is hit and miss whether or not those methods will be permitted for any given transaction. Asking the seller for a pro-forma invoice (PI), then heading to the bank to send a wire, and trusting to their honesty should work just fine when dealing with companies that have a long-standing gold badge.

To enable shipping with tracking via carriers such as DHL, the preferred method of delivery for Chinese suppliers shipping to the US or Europe, you will need to provide a shipping address, email address, and phone number. Those details will find their way into spam databases if you are dealing with more than a few companies, and will be, of course, sold on by Alibaba itself as well. Expect to see an uptick of spam after dealing with suppliers via Alibaba, so consider using throwaway credentials where possible.

Chinese manufacturers active on Alibaba are familiar with international shipping practices. On their own initiative may or may not decide to declare the true cost and contents of the shipped package. This is another form of widely practiced civil disobedience, but is much more common in the shipping of pharmaceuticals than in the shipping of synthesized proteins. The former are likely to be confiscated by customs officials, while the latter are not. If the true cost is declared, then expect to pay customs duty on that cost; payment is typically handled via the carrier. Note that different carriers tend to have different processes and rates at which shipments are checked for validity.

Storing Flagellin

Proteins are shipped in a solid freeze-dried (lyophilised) form. While in this form they are easily stored in a refrigerator for the short-term or in a freezer for the long term. It has a much shorter life span once it has been mixed with liquid for injection, however, and should be kept frozen, and used within a matter of a few months at the most.

Validating the Purchased Flagellin

A protein may have been ordered, but that doesn't mean that what turns up at the door is either the right one or free from impurities or otherwise of good quality. Even when not ordering from distant, infrequent suppliers, regular testing of batches is good practice in any industry. How to determine whether a protein is what it says it is on the label? Run it through a process of liquid chromatography and mass spectrometry, and compare the results against the standard data for a high purity sample of that compound. Or rather pay a small lab company to do that.

Obtain an Extra Vial from the Same Batch as the Others

Since it is extra work to attempt to split out microgram amounts of protein or mail protein in solution rather than lyophilised protein, just order an extra vial from the same batch and send it off to be tested.

Use Science Exchange to Find Lab Companies

Science Exchange is a fairly robust way to identify providers of specific lab services, request quotes, and make payments. Here again, pick a small lab company to work with after searching for LC-MS (liquid chromatography and mass spectrometry) services. Large companies will want all of the boilerplate registrations and legalities dotted and crossed, and are generally a pain to deal with in most other ways as well. Companies registered with Science Exchange largely don't provide their rates without some discussion, but a little over $100 per sample is a fair price for LC-MS to check the identity and purity of the compound.

Work with the Company to Arrange the Service

The process of request, bid, acceptance, and payment is managed through the Science Exchange website, with questions and answers posted to a discussion board for the task. Certainly ask if you have questions; most providers are happy to answer questions for someone less familiar with the technologies used. Service providers will typically want a description of the compounds to be tested and their standard data sheets, as a matter of best practice and safety. Here provide the mass spectra and other data sheets from the vendor, or use those published by NovoPro or other sources.

Ship the Samples

Ship the sample via a carrier service such as DHL, UPS, or FedEx. Some LC-MS service companies may provide shipping instructions or recommendations. These are usually some variety of common sense: add a description and invoice to the package; reference the order ID, sender, and receiver; clearly label sample containers; and package defensively with three layers of packing; and so forth.

Examine the Results

Once the LC-MS process runs, the lab company should provide a short summary regarding whether or not the compound is in fact the correct one and numbers for the estimated purity. Also provided are the mass spectra, which can be compared with the existing spectra from the vendor or other sources.

Establishing Tests and Measures

There are a few options for testing before and after, the most compelling of which is the gut microbiome. Changes there, given a stable diet, should be indicative that something happened. But to assess outcomes beyond that, inflammatory markers should be tested at the very least. It might also be interesting to look at DNA methylation assessments of biological age, though it is an open question as to what exactly these tests are measuring, and whether they are all that useful to an individual.

Gut Microbiome

The most direct measure of effectiveness is to assess the gut microbe populations directly. There are lab services that do this, but few commercial direct to consumer services. One US based service is Viome, though it doesn't present results in a useful tabular form, and does not provide the raw data. Nonetheless, it is a measurable endpoint. Gut microbe populations are fairly slow to change, given a consistent diet, so large differences over a ten week course of flagellin injections would be interesting to observe.

Bloodwork

There exist online services such as WellnessFX where one can order up a blood test and then head off the next day to have it carried out by one of the widely available clinical service companies. Since inflammatory markers are the topic of interest, a service that offers more individual tests rather than packages might be a better choice. The Life Extension Foundation offers a wide range of tests, for example, including a selection of inflammatory markers.

DNA Methylation

DNA methylation tests can be ordered from either vendors such as Epimorphy / Zymo Research - note that it takes a fair few weeks for delivery. From talking to people at the two companis, the normal level of variability for repeat tests from the same sample is something like 1.7 years for the Zymo Research test. The level of day to day or intraday variation between different samples from the same individual remains more of a question mark at this point in time, though I am told they are very consistent over measures separated by months. Nonetheless, it is wise to try to make everything as similar as possible when taking the test before and after a treatment: time of day, recency of eating or exercise, recent diet, and so forth.

Guesstimated Costs

The costs given here are rounded up for the sake of convenience, and in some cases are blurred median values standing in for the range of observed prices in the wild. The choice to use needles for subcutaneous injection is obviously much cheaper than exploring the world of needle-free injections and vial assembly.

• Business mailbox, such as from UPS: $250 / year
• Cytokine blood panel test from LEF: $300 / test
• MyDNAage kits: $310 / kit
• Viome kits: $150 / kit
• Miscellenous equipment: spatulas, labels, vials, a vial rack, etc: $60
• Small pack of 13mm sterile serum vials: $35
• PharmaJet Needle-free Injection Kit: $1020
• Comfort-in Needle-free Injection Kit: $470
• Bulk 13mm serum vial parts and capping tools: $750
• 100 μg of flagellin via Alibaba: $1000
• Shipping and LC-MS analysis of a sample: $200

Practice Before Working with Flagellin

Do you think you can reliably pipette fluid in 0.5ml amounts between small vials? Or cap vials or connect adaptors or fill syringes or carry out an injection without messing it up somewhere along the way? Perhaps you can. But it is a very good idea to practice first with saline solution rather than finding out that your manual dexterity and methods are lacking while handling an expensive protein. You will doubtless come to the conclusion that more tools or different tools are needed than was expected to be the case.

Schedule for the Self-Experiment

One might expect the process of discovery, reading around the topic, ordering materials, and validating an order of flagellin to take a couple of months. Once all of the decisions are made and the materials are in hand, pick a start date. The schedule for the self-experiment is as follows:

• Day 0: Perform the various tests: bloodwork, gut microbiome assessment, etc.
• Day 1: Intramuscular injection of 10 μg of flagellin.
• Day 8: Intramuscular injection of 10 μg of flagellin.
• Day 15: Intramuscular injection of 10 μg of flagellin.
• Day 22: Intramuscular injection of 10 μg of flagellin.
• Day 29: Intramuscular injection of 10 μg of flagellin.
• Day 36: Intramuscular injection of 10 μg of flagellin.
• Day 43: Intramuscular injection of 10 μg of flagellin.
• Day 50: Intramuscular injection of 10 μg of flagellin.
• Day 57: Intramuscular injection of 10 μg of flagellin.
• Day 64: Intramuscular injection of 10 μg of flagellin.
• Day 65: Repeat the tests.

Where to Publish?

If you run a self-experiment and keep the results to yourself, then you helped only yourself. The true benefit of rational, considered self-experimentation only begins to emerge when many members of community share their data, to an extent that can help to inform formal trials and direction of research and development. There are numerous communities of people whose members self-experiment with various compounds and interventions, with varying degrees of rigor. One can be found at the LongeCity forums, for example, and that is a fair place to post the details and results of a personal trial. Equally if you run your own website or blog, why not there?

When publishing, include all of the measured data, the doses taken, duration of treatment, and age, weight, and gender. Fuzzing age to a less distinct five year range (e.g. late 40s, early 50s) is fine. If you wish to publish anonymously, it should be fairly safe to do so, as none of that data can be traced back to you without access to the bloodwork provider. None of the usual suspects will be interested in going that far. Negative results are just as important as positive results! Publish whatever the outcome.