Maybe you are about to start working with D-luciferin, and you begin reviewing protocols. But what you’re doing is a little different. Or what you are using requires a different amount of D-luciferin. There are standard protocols out there that define how much solvent to use when you have a given amount of D-luciferin. But not every experiment is the same. Therefore, this is just a quick refresh on how to do some easy lab math to figure out how much solvent you would need to use when reconstituting your lyophilized D-luciferin.

The steps to calculating how much solvent to use for D-luciferin reconstitution involve knowing your amount of luciferin needed and your desired concentration, making proper unit conversions, and dividing out the numbers.

This is an oversimplified answer though. So, we’ll go through it step by step.

 

Table of Contents

1.     Write down your “givens”

2.     Convert your units (if needed).

3.     Use the relationship between your mass and concentration

4.     Plug your numbers into the formula

Trying an example

Important considerations

Storage considerations

Aliquoting D-luciferin after reconstitution

Equipment Needed

References

 

 

1.    Write down your “givens”

Write down your knowns or your givens. This will be the amount of luciferin you plan to use and the desired concentration.

For our example, we’ll say we’re working with 1 gram of lyophilized D-luciferin, and we need a 15 mg/mL concentration. Our givens would be:

Givens:

·         Mass of luciferin = 1 g

·         Desired concentration = 15 mg/mL

 

2.    Convert your units (if needed).

In our example, we’re working with 1 gram of D-luciferin, and our desired concentration is 15 mg/mL. To do the math right, we need our units of mass to be uniform. In this case, we want to convert everything to milligrams.

That is easy enough: 1 gram of D-luciferin = 1,000 mg of D-luciferin.

So now we’re looking at our list like this:

·         Mass of luciferin = 1,000 mg

·         Desired concentration = 15 mg/mL

 

 

3.    Use the relationship between your mass and concentration

The concentration of something is its mass over its volume. As an equation, that looks like this:

Concentration = (Mass) / (Volume)

The problem is, volume is what we’re trying to figure out. So, we can rearrange the equation:

Volume = (Mass) / (Concentration)

 

4.    Plug your numbers into the formula

We’re at the easy part of this whole thing. Actually, it was all pretty easy. This might be the more satisfying part where we just plug in our numbers and solve.

Volume = (1,000 mg) / (15 mg/mL)

Volume = 66.7 mL

 

Remember with fractions, if you have the same number on the top and on the bottom, they can cancel out. The same is true for units. If you look at your equation above, you have mg on the top and on the bottom. When you cancel out those two units, you’re left with mL as your only remaining unit, confirming you’ve solved for your needed volume.

 

Answer:

To make a 15 mg/mL solution from 1 g luciferin, add 66.7 mL of solvent (either sterile water or PBS).

 

Trying an example

To test it all out, let’s try with a different example. Let’s say you’re working with 500 mg of D- luciferin, and you want a 30 mg/mL concentration.

1.    Start with your givens:

o   Mass: 500 mg D-luciferin

o   Concentration: 30 mg/mL

2.    Conversion: You don’t need to do any unit conversion in this example because everything is in milligrams already. We get to move to the next step.

3.    Prepare your formula: Volume = Mass/Concentration

4.    Plug in the values and solve: Volume = (500 mg) / (30 mg/mL)

5.    Answer: 16.67 mL solvent.

 

Important considerations

When mixing, there is no need for aggressive vortexing. Instead, mix by gentle inversion or slow pipetting up and down. The D-luciferin potassium or sodium salt should readily dissolve (Figure 1). Please note, we are not talking about the free acid form of D-luciferin, which requires other steps to help it dissolve.

Figure 1. Illustrates inversion, which is rotating the bottle in your hand up and down to gently mix material.

If the solution will be used in cell culture, sterile technique is essential. Prepare the solution using sterile water or PBS in a biosafety cabinet using aseptic practices. For in vivo applications, particularly injection into animals, sterile filtration through a 0.22 µm filter is required. In these cases, also use sterile, endotoxin-free water and sterile containers, and be aware that endotoxin contamination can impact results and harm animals. Use endotoxin-free reagents and consumables where possible.

 

Storage considerations

It is important to aliquot your stock and freeze it immediately. For long-term storage, store your single-use aliquots at −80°C and protect them from light. When properly stored, your reconstituted D-luciferin should be stable for up to 6 months.

For working stock solutions, keep it at 4°C and protected from light for up to 1-2 days. On the day of your experiment, prepare your dilute working solution (from your thawed aliquoted stock solution) and use it immediately.

 

Aliquoting D-luciferin after reconstitution

Once you’ve reconstituted your D-luciferin, the clock starts ticking when it comes to stability. Freezing any stock helps slow degradation. But repeatedly thawing the same tube quickly lowers its stability. This is why aliquoting your stock is so important.

The goal of aliquoting is to divide your stock into volumes that match your single experimental use. For example, if you were doing assays that required 200 µL of 15 mg/mL D-luciferin stock, you would want to divide your stock into tubes that are each filled with 200 µL of your stock. This way your tubes only go through a single thawing cycle, leaving the rest of your stock (now divided) happy in the -80°C freezer.

Figure 2. Concentrated luciferin stock (15 mg/mL) is aliquoted into appropriate tubes in single-use volumes.

 

When aliquoting, you want to stay very organized and label each tube very clearly with what the material is, the concentration, date of preparation, and your initial if applicable. This is particularly important in labs where multiple researchers share reagents. Accurate labeling prevents uncertainty about storage duration or preparation conditions.

You’ll want to store your luciferin protected from light. For single-use aliquots you can throw them in a freezer box to shield the light when you and your colleagues repeatedly open the freezer. For the stock solution you can wrap the bottle in aluminum foil to minimize the light shining on your luciferin stock.

Lastly, it is important to maintain sterility throughout the whole aliquoting process.

 

 

Equipment Needed

Now that you know your volume, and how to store things safely, you are probably feeling ready to prepare your luciferin solution. But that could also leave you questioning what to use to mix your luciferin stock in the first place and what you should aliquot everything into. Here is a quick supply list.

Weighing and Measuring:

·         Analytical balance

·         Clean weigh boats

·         Spatula or scoop

Preparation Vessels

·         Sterile 100 mL glass bottle with cap

Reagents:

·         D-luciferin potassium or sodium (lyophilized)

·         Endotoxin-free D-luciferin potassium or sodium (recommended for in vivo)

Solvent

·         Sterile water or sterile PBS

·         Serological pipette or graduated cylinder for measuring out your solvent

Sterile Filtration

·         0.22 µm vacuum filtration unit for sterilizing larger volumes

·         0.22 µm syringe filter with syringe for smaller volumes

·         Biosafety cabinet for maintaining aseptic technique throughout filtration and aliquoting

Aliquoting

·         Single channel pipette with sterile tips for transferring stock into individual aliquot tubes

·         Cryovials or centrifuge tubes for long-term storage at −80°C.

·         Aluminum foil to protect stock solutions from light

·         Tube rack

·         Permanent marker for tube labeling

·         Freezer box for aliquots

Personal Protective Equipment

·         Nitrile gloves

·         Lab coat

·         Eye protection

 

 

References

De Almeida, P. E., van Rappard, J. R., & Wu, J. C. (2011). In vivo bioluminescence for tracking cell fate and function. American Journal of Physiology-Heart and Circulatory Physiology, 301(3), H663-H671.

Gao, C., Wu, Y., Fitzgerald, C., Wang, H., Ugo, T., Uyeda, T., ... & Lin, M. Z. (2025). An optimized luciferin formulation for NanoLuc-based in vivo bioluminescence imaging. Scientific Reports, 15(1), 12884.

Gold Biotechnology. (n.d.). In vivo luciferin FAQs. https://goldbio.com/articles/article/in-vivo-luciferin-faqs

Kuchimaru, T., Iwano, S., Kiyama, M., Mitsumata, S., Kadonosono, T., Niwa, H., ... & Kizaka-Kondoh, S. (2016). A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging. Nature communications, 7(1), 11856.

 

 

 

 

 

 

 

 

 

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