Protocol for preparing monosodium urate crystals for in vitro and in vivo studies in mouse and human cells

Summary

Gout is caused by the deposition of monosodium urate crystals (MSUc) in the joints, triggering a unique inflammatory and metabolic response in macrophages. Here, we present a protocol to generate MSUc for in vitro and in vivo studies in mouse and human cells. We describe steps for dissolving uric acid followed by crystallizing, purifying, evaluating, and analyzing MSUc. We then detail procedures for stimulating human/mouse-derived macrophages and determining endotoxin levels in MSUc preparation.

Graphical abstract

Highlights

• •Dissolving uric acid to initiate MSUc formation
• •Crystallization process for needle-shaped MSUc
• •Purification and evaluation of MSUc purity
• •Stimulating macrophages and assessing endotoxin levels

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Gout is caused by the deposition of monosodium urate crystals (MSUc) in the joints, triggering a unique inflammatory and metabolic response in macrophages. Here, we present a protocol to generate MSUc for in vitro and in vivo studies in mouse and human cells. We describe steps for dissolving uric acid followed by crystallizing, purifying, evaluating, and analyzing MSUc. We then detail procedures for stimulating human/mouse-derived macrophages and determining endotoxin levels in MSUc preparation.

Before you begin

This protocol presents a detailed explanation of how to generate monosodium urate crystals (MSUc). While other approaches allow to produce large amounts of uric acid crystals, the temperature, pH in the formation of sodium hydroxide must be carefully monitored to avoid formation of large MSUc, which can compromise the downstream application. In contrast, our protocol offers a robust and reproducible way to produce MSUc with a needle-shaped structure that is characteristic of patients with gout, that can be used for in vitro and in vivo experiments.

Institutional permissions

All animal experiments were performed in agreement with the Institutional Animal Care and Use Committee (IACUC). The readers must obtain authorization from the appropriate institution to conduct animal model experiments.

Sterilization of lab utensils, equipment calibration and preparation of solutions

Timing: 2 h

• 1.
  Sterilization of the lab utensils is crucial to avoid contamination with other substances. For this:

  • a.
    Wash the following lab utensils thoroughly with double distilled water- 1 L glass beaker, laboratory scoop, micro spatulas, and 1 magnetic stirrer.
  • b.
    Wrap the lab utensils in aluminum foil and autoclave for 30 min at 250°F (121°C) and 15 min at 270°F (132°C), with a dry time of 20 min.
  • c.
    Let the utensils cool down before use.
• 2.Calibration of pH meter should follow the manufacturer’s recommendations (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLPD%2FTechnical-Notes%2FTechNote-LSTAR1117-pH-BenchMeter.pdf).
• 3.
  Prepare 1 M NaOH in ultrapure distilled water as follows:

  • a.
    Add 2 g of NaOH for every 50 mL of ultrapure distilled water and mix until completely dissolved.

Preparation of uric acid

Timing: 15 min

• 4.Weigh 5 g of uric acid crystals using a weigh boat or aluminum foil.

Note: Uric acid is toxic and must be handled with care.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
200 Proof pure ethanol	Koptec	2716GEA
DMEM	Gibco	5796
DPBS no calcium, no magnesium	Gibco	14190-144
EDTA	Invitrogen	AM9262
FBS	Biowest	S1650
HBSS	Gibco	14175-095
Human recombinant MCSF	STEMCELL Technologies	78057-2
Mouse recombinant MCSF	BioLegend	576406
Pen/Strep	Gibco	10378016
RPMI	Sigma-Aldrich	10–040
Sodium hydroxide	Sigma	S5881-500G
UltraPure distilled water	Invitrogen	10977015
Uric acid	Sigma	U0881-25G
Other
10 mL syringe	Luer-Lock	302995
50 mL tubes	Fisher Scientific	14-432-22
Balance	Mettler Toledo	ME104E
Beaker	PYREX	CLS1000600
Buchner funnel	Deschem	X002Q5NO7T
Eppendorf tubes	LabForce	1149X63
Filter Stericup	Millipore	S2GPU11RE
Filtering paper (110 mm pores)	ZenPore	ST002-110
Flask	Fisher Scientific	FB500500
Glass bottle	Karter Scientific	251M4
Hexagonal weighing dish	Fisherbrand	02-202-102
Hot plate	Fisher Scientific	11-300-49SHP
Needle 18G	BD	305196
Needle 22G	BD	305156
Needle 30G	BD	305128
pH meter	Fisher Scientific	13-636-AB200A
Rubber adapters	StonyLab	BI-9PK-GX
Spoonulet	Fisher Scientific	14-375-20
Vacuum tubing	Thermo Scientific	13-713-01

Materials and equipment

Mouse Bone Marrow-Derived Macrophage Media

Reagent	Final concentration	Amount
DMEM	N/A	450 mL
Fetal Bovine Serum (heat inactivated) (100%)	10%	50 mL
Penicillin/Streptomycin	100 U	5 mL
Murine MCSF (50 μg/mL)	16–25 ng/mL	8–12.5 μg
Total	N/A	500 mL

Note: Although the media can be stored at 4°C for a maximum storage time of 6 months, MCSF is added fresh when starting to differentiate the mouse or human macrophages.

Human Monocyte-Derived Macrophage Media

Reagent	Final concentration	Amount
RPMI	N/A	450 mL
Fetal Bovine Serum (heat inactivated) (100%)	10%	50 mL
Penicillin/Streptomycin	100 U	5 mL
Human MCSF (50 μg/mL)	16–25 ng/mL	8–12.5 μg
Total	N/A	500 mL

Note: Although the media can be stored at 4°C for a maximum storage time of 6 months, MCSF is added fresh when starting to differentiate the mouse or human macrophages.

HBSS Media

Reagent	Final concentration	Amount
HBSS	N/A	450 mL
Fetal Bovine Serum (heat inactivated) (100%)	10%	50 mL
EDTA (0.5 M)	1 mM	0.5 mL
Total	N/A	500.5 mL

Note: The media can be stored at 4°C for a maximum storage time of 6 months.

Step-by-step method details

Dissolve uric acid in water

Timing: 4 h

This step aims to dissolve the uric acid in water while setting up the solution to an appropriate temperature.

• 1.Pour the previously measured 5 g of uric acid crystals into an empty beaker with a magnetic stir bar.

Note: that Uric acid is toxic and must be handled with care.

• 2.Place beaker on the hot plate.

Note: The time required to reach 60°C can indeed vary significantly depending on the specific model and efficiency of the hot plate being used. Therefore, we do not specify exact settings, as they might not be universally applicable. We recommend starting with a medium heat setting and closely monitoring the temperature until you become familiar with how quickly your hot plate reaches the desired temperature. Adjust the settings as necessary to maintain the target temperature for the required duration. Always ensure to use a reliable thermometer to monitor the temperature accurately.

• 3.
  Add 650 mL of Ultrapure Distilled Water to the beaker.

  • a.
    Increase the heat to 60°C and start the magnetic stirrer with 400 × g.
• 4.
  Place the thermometer into the beaker to monitor temperature ensuring that the thermometer is not touching the glass beaker or stir bar.

  • a.
    Aim for thermometer to reach 60°C.

Note: Because of the large amount of liquid, the heating process can take upwards to 4 h to get to 60°C

• 5.
  Wash the pH meter with distilled water and place the meter into the beaker to monitor the pH.

  • a.
    Cover the top of the beaker with aluminum foil.

    Note: While minor water evaporation is not expected to significantly impact the final outcome, covering the beaker can help minimize evaporation and ensure more consistent results.

  • b.
    Note down the pH every 30 min.

    Note: As the uric acid dissolves from the increase in heat, the pH will continue to decrease.

    Note: Once pH does not change more than +/− 0.1 pH and temperature is ∼60°C, indicates that the uric acid solution has saturated.

    Note: At the stage, the solution will look cloudy, milky, and white with much solute not dissolved. Troubleshooting 1.

Crystallization of uric acid into monosodium urate crystals

Timing: 16 h

This step aims to produce monosodium urate crystals with a needle-like shape to resemble the crystal found in the synovial fluid in gout patients.

• 6.Add 1 mL of 0.1 M NaOH at a time to the beaker (Methods video S1).
• 7.
  Write down pH after pH stabilizes on the pH meter.

  Note: As more NaOH is added, the solution will slowly become more transparent, less cloudy

  • a.
    Repeat steps 6 and 7 until adding the 1 mL of NaOH results in the pH to spike ∼1 pH unit after stabilizing (critical point).

    Note: Around 11–14 mL of NaOH needs to be added before a noticeable pH spike will happen. Critical point is reached when pH is around 7.5. (Methods videos S2 and S3)

    Note: For reference, the critical point will reach when pH is around 7.50 (https://drive.google.com/file/d/1G90aR8UPQWPvIrTp-Lr3-eqAWzlg0M-d/view?usp=sharing).
• 8.Add one more 1 mL of NaOH.

Methods video S1. Example of how adding 1M NaOH fluctuates pH before critical point, related to step 6

(https://drive.google.com/file/d/1vjYGgEq7RZNFhgSj9foPJq86qWvkqcRn/view?usp=sharing).

Example of pH trend

1 M NaOH added (mL)	pH after stabilizing	Comments
0	3.75	Saturation point
1	5.63
2	5.98
3	6.16
4	6.30
5	6.40
6	6.48
7	6.55
8	6.60
9	6.67
10	6.75
11	7.50	Critical point
12	8.76	Significant jump in pH

• 9.Turn off the heat and magnetic stirrer.
• 10.
  Take beaker off the hot plate and let it sit. Troubleshooting 2.

  • a.
    Crystals will slowly start to form as temperature decreases – (Figure 1 and Methods video S4).
  • b.
    Let the beaker sit 12–16 h on bench top for crystallization process to complete.

Figure 1.

Crystals forming after heat is turned off

This image shows the formation of crystals once the solution reaches the appropriate pH and temperature. Please note the consistency of the crystals to avoid confusion with uric acid precipitation due to poor crystallization.

Methods video S4. One hour time-lapse of crystallization process after removing heat, related to step 10

(https://drive.google.com/file/d/1T1TwEsXJZiZbVfGU7PustQ_7-jXEfoyQ/view?usp=sharing).

Wash, clean and dry monosodium urate crystals

Timing: 5 h

This step aims to purify monosodium urate crystals to make them suitable for in vivo and in vitro studies.

• 11.Transfer all the crystals from the beaker into 50 mL tubes with each tube containing 40 mL of crystal mixture.
• 12.Centrifuge at 2000 × g for 5 min to separate the liquid and aspirate liquid (Figure 2).
• 13.Wash each 50 mL tube of crystals with 20 mL of 75% EtOH once (dilute 100% EtOH with sterile water) by pipetting up and down the mixture to mix the 75% EtOH.
• 14.Centrifuge to separate liquid and aspirate it.
• 15.Wash each 50 mL tube of crystals once with 20 mL sterile PBS by pipetting up and down the mixture to mix the sterile PBS.
• 16.Centrifuge to separate liquid and aspirate it.
• 17.
  Vacuum Filtrate the liquid from the crystals to begin the drying process.

  • a.
    Option 1: Use the Millipore Filter Stericup (Figure 3).
  • b.
    Option 2: Set up a regular vacuum filtration system with a side-arm flask, Büchner funnel with filter paper, and vacuum trap.
  • c.
    After vacuum filtration, the filtration system is opened, and the filter paper with crystals is carefully collected using a cell scraper.

Note: At this point, there will still be moisture left – the crystals will be pasty. During the collection of crystals after vacuum filtration, it is important not to disrupt the membrane of the filtration system. We suggest to employ a cell scraper for carefully collecting the crystals from the filter paper, which ensures the integrity of the filter membrane is maintained while efficiently retrieving the crystals for subsequent steps.

• 18.Spread crystals onto a broad surface of aluminum foil and bake at 80°C. Troubleshooting 3.

Note: before baking the crystals, we recommend thoroughly cleaning the incubator with bleach to ensure a sterile environment. Additionally, avoid using an incubator that was previously used for bacteria culture to prevent contamination. Ensuring these precautions will aid in maintaining the purity and integrity of the crystals throughout the baking process.

• 19.Resuspend crystals in fresh sterile DPBS (2–3 mg/mL).

Note: MSUc can be stored at 4°C for up to 6 months and at −20°C for up to 1 year, ensuring their stability and usability for future experiments.

Figure 2.

Crystals after centrifugation per step 12

This image displays the crystals after centrifugation. As in Figure 1, observe the consistency of the crystals.

Figure 3.

Example of a Millipore filter Stericup setup for vacuum filtration of crystals

To minimize the chances of contamination with endotoxins, vacuum filtration is performed inside a tissue culture hood.

Microscopic evaluation and analysis of monosodium urate crystals

Timing: 2 h

This step aims to evaluate the size, shape, and consistency of monosodium urate crystals.

• 20.Obtain a 100 μL aliquot of the MSU crystals.
• 21.Place aliquot on a microscope slide.
• 22.
  Visualize the crystal preparation under microscope.

  • a.
    Crystals should have needle like shape (Figure 4) Troubleshooting 4.
• 23.Conduct RNA expression analysis (RT-qPCR or RNA Seq) of LPS-induced genes to check for LPS contamination in the MSU crystals.

Note: The steps involving the use of needles must be done with care.

Figure 4.

Needle-like structure of crystals after the crystallization process

This image shows the needle-like structure of the crystals, serving as a quality control measure for the final product.

Stimulation of mouse bone marrow-derived macrophages and human monocyte-derived macrophages by monosodium urate crystals

Timing: 2–5 h

This step aims to optimize the delivery of monosodium urate crystals to mouse and human macrophages for in vitro experiments. Troubleshooting 5.

• 24.
  Extract bone marrow from femurs and tibias of wild-type mice (option 1) or isolate human peripheral blood mononuclear cells from whole blood (option 2).

  • a.
    Culture in DMEM/RPMI medium supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin, 2 mM L-glutamine, and 10 ng/mL recombinant murine M-CSF (option 1, for murine BMDM) or 25–50 ng/mL recombinant human M-CSF (option 2, for human MDM) at 37°C and 5% CO₂ for six days.
  • b.
    On day six, replate the cells with the DMEM/RPMI.
  • c.
    One day after being in the fresh media, stimulate the cells for 5 h with 250 μg/mL of MSUc.
  • d.
    Isolate RNA for quantitative PCR or RNA-Seq to check the expression of classical endotoxin-induced genes as read-out of endotoxin contamination (Figure 5).

Note: For detailed description on the protocol to generate of mouse bone marrow-derived macrophages and human monocyte-derived macrophages, please refer to Refer to Cobo et al.^1,2 and Toda et al.³

Note: To cultivate bone marrow-derived macrophages (BMDM), we recommend flushing the bone marrow cells from the femurs and tibias of mice. This process involves carefully extracting the bone marrow using a syringe filled with DMEM medium supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin, 2 mM L-glutamine, and 10 ng/mL recombinant murine M-CSF, ensuring that the cells are collected efficiently.

Note: To cultivate human monocyte-derived macrophages, we recommend obtaining the PBMC using a Ficoll-gradient method, which separates the blood cells based on their density. The PBMC layer, located at the interface of the density gradient, is carefully collected and monocytes are isolated using a negative-selection kit to avoid disrupting the cells.

Note: We recommend using DMEM to grow BMDM and RPMI to grow MDM.

Note: In our experience, we recover 35–40 M cells from an adult (8–12 weeks old) male and 25–30 M from an adult female.

Note: We recommend seeding the cells at a density of 1 M cells/ml of media. We recommend not to change the media during the six-day differentiation phase. Our method is tailored to sustain the cells for this duration with no additional media changes needed. It is crucial not to disturb the cultures during the differentiation.

Note: We recommend culturing cells in a 12-well plate, seeding 1 million (1 M) cells per well. For experiments utilizing a 6-well plate, the seeding density should be increased to 2 million (2 M) cells per well. This adjustment in cell density ensures that each well has a sufficient number of cells to respond to the MSUc , taking into account the differing surface areas of the wells in a 12-well and a 6-well plate.

Note: We find real time quantitative PCR or RNA-Seq more sensitive than other ELISA-based methods to detect endotoxin contamination. For details about the protocol to stimulate macrophages with MSUc, and the source of data in Figure 5, refer to Cobo et al.¹

Figure 5.

Expression of endotoxin-induced genes by RNA-Seq (TPM denotes Transcripts Per Million Reads) (n = 3/group)

The graphs depict the expression of Mx1, Mx2, Oasl1, and Ifit2 in mouse bone marrow-derived macrophages (BMDMs) stimulated with 100 ng/mL of LPS or 250 μg/mL of MSUc for 5 h. Error bars denote the Standard Error of the Mean. ∗ P<0.05; ∗∗ P<0.01Data obtained from Cobo et al.¹

Expected outcomes

By following the presented protocol, one should expect to generate monosodium urate crystals with a characteristic needle shape. When added to macrophages, they phagocytose the crystals and initiate an inflammatory response.

Limitations

While highly useful in various research and diagnostic applications, the protocol for producing monosodium urate crystals is subject to several limitations that may affect the reliability and validity of the results. One significant limitation is the potential to contaminate the uric acid crystals with other substances, which can induce precipitation but inhibit proper dissolution and lower crystal quality formation. Moreover, the protocol’s success is highly dependent on the precise optimization of temperature and time parameters for dissolving the uric acid powder. Inadequate control over these conditions can lead to incomplete dissolution, affecting crystal formation and morphology. Uric acid crystals are known to form rapidly under certain conditions, but without careful control, they may not acquire the characteristic needle shape, which is essential for their identification and utilization in specific applications to mimic the clinical scenario of gouty inflammation. Another critical consideration is that the crystals might contain traces of endotoxin, which can be a significant concern in biomedical applications where the presence of endotoxin can influence experimental outcomes or trigger unwanted biological responses. Environmental factors, such as humidity and ambient temperature, mechanical limitations, the precision of temperature control equipment, and the homogeneity of mixing during dissolution, can further affect the protocol’s efficacy and the integrity of the monosodium urate crystals produced. To ensure the reproducibility of the protocol, maintaining sterile conditions for all materials used and optimizing the crystallization time are crucial steps. We recognize that there will be some variation between different crystal preparations; thus, to minimize experimental variability, we recommend using crystals from the same lot for the entirety of a project. This approach helps in achieving consistent results and enhances the reliability of your findings.

Troubleshooting

Problem 1

Solution does not turn milky and shows precipitates.

Related to Step 4.

Related to Step 5b.

Potential solution

• •Increase the temperature to 65°–70°C.
• •Add 100 mL more sterile water.

Problem 2

Crystallization does not occur at ∼7.5 pH.

Related to Step 10.

Potential solution

• •Stop the heater and wait for at least 10 min to see if crystals start forming slowly.

Note: For additional information on the nucleation of monosodium urate crystals, please refer to Wilcox and Khalaf, 1975).⁴

Problem 3

Crystals show rock-solid structure.

Related to Step 17.

Potential solution

• •Do not over dry the crystals.
• •Use a rather broad surface for drying.
• •Crystals should not look pasty but should not be over dry.
• •Unfortunately, if the crystals show a rock-solid structure, we recommend to start the protocol all over again.

Problem 4

Some crystals do not have the needle-like shape.

Related to Step 21.

Potential solution

• •Try to run the crystal solution through a 18G-30G needle.
• •There is a chance that crystallization did not work properly.

Problem 5

Low cell viability at 5–8 h after stimulation with MSUc.

Related to Step 22.

Potential solution

• •Titrate [MSUc]. Effect of MSUc in macrophages depends on the degree of terminal differentiation of macrophages.
• •For longer time points, remove the media and add fresh one after 5 h.
• •Shake MSUc before use to ensure proper dissolution.

Resource availability

Lead contact

Isidoro Cobo (isidorocobo@uabmc.edu).

Technical contact

Mohnish Alishala (malishal@ucsd.edu).

Materials availability

This study did not generate unique reagents.

Data and code availability

The protocol includes representative images used during the study. Original images with improved resolution are available upon request to the lead author. This study did not generate datasets/codes.