Protocol for measuring endogenous 24(S),25-epoxycholesterol and inducing innate immune memory in mouse macrophages

TL;DR

This paper provides a detailed protocol for measuring a specific cholesterol metabolite and inducing immune memory in mouse macrophages.

Contribution

A new protocol for quantifying 24(S),25-epoxycholesterol and inducing trained immunity in mouse macrophages is introduced.

Findings

A workflow for precise quantification of 24(S),25-epoxycholesterol using LC-MS is described.

Procedures for establishing metabolite-induced trained immunity in BMDMs are detailed.

ATAC-seq and cytokine assays are outlined for evaluating immune memory.

Abstract

Multiple metabolic pathways and metabolites are involved in innate immune memory induction of macrophages; however, protocols for in vitro-trained immunity assays induced by metabolites in mouse macrophages are limited. Here, we present a protocol for measuring endogenous 24(S),25-epoxycholesterol and inducing innate immune memory in mouse macrophages. We describe steps for sample preparation, measurement of 24(S),25-epoxycholesterol, and establishment of an in vitro-trained immunity model. We then detail procedures for assays measuring cytokine concentration and for assay for transposase-accessible chromatin using sequencing (ATAC-seq).

For complete details on the use and execution of this protocol, please refer to Liu et al.1

•Instructions on culturing and differentiation of bone marrow-derived macrophages (BMDMs)•Workflow for precise quantification of endogenous 24(S),25-EC using LC-MS•Validation procedures for establishing metabolite-induced trained immunity in BMDMs

Instructions on culturing and differentiation of bone marrow-derived macrophages (BMDMs)

Workflow for precise quantification of endogenous 24(S),25-EC using LC-MS

Validation procedures for establishing metabolite-induced trained immunity in BMDMs

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

Multiple metabolic pathways and metabolites are involved in innate immune memory induction of macrophages; however, protocols for in vitro-trained immunity assays induced by metabolites in mouse macrophages are limited. Here, we present a protocol for measuring endogenous 24(S),25-epoxycholesterol and inducing innate immune memory in mouse macrophages. We describe steps for sample preparation, measurement of 24(S),25-epoxycholesterol, and establishment of an in vitro-trained immunity model. We then detail procedures for assays measuring cytokine concentration and for assay for transposase-accessible chromatin using sequencing (ATAC-seq).

Figures

Gating strategy of *in vitro* differentiated BMDMs (Zombie^−^ F4/80^+^ CD11b^+^)

Measurement of endogenous 24(S),25-EC in BMDMs(A) The peak area of gradient-diluted standards and samples.(B) The standard curve for the measured 24(S),25-EC standards.(C) Endogenous level of 24(S),25-EC in BMDMs. Mean ± SEM with five independent samples.Figure 3Measurement of intracellular 24(S),25-EC with addition of exogenous 24(S),25-EC(A) The measured values of the CCK-8 assay in BMDMs treated with various concentrations of 24(S),25-EC for 24 h.(B) The normalized OD450 of the CCK-8 reaction assay in BMDMs. Mean ± SEM with five independent samples, by one-way ANOVA test followed by Tukey’s

ELISA assay measuring TNF-α, IFN-β, and IFN-γ in the supernatants from 24(S),25-EC-trained BMDMs(A) The standard curve of TNF-α, IFN-β and IFN-γ standards.(B) The supernatant levels of TNF-α, IFN-β and IFN-γ in trained BMDMs after LPS restimulation. Mean ± SEM with three independent experiments, by two-way ANOVA test followed by Sidak’s multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.001.

ATAC-seq assay measuring the chromatin accessibility of 24(S),25-EC-trained BMDMs(A) AGE of amplified DNA libraries with different amplifying cycles.(B) Density plots and heatmaps of all peaks depict the global chromatin accessibility around TSS regions in each group. Kb stands for kilobase and TSS stands for transcription start site. WGP serves as a positive control for inducing innate immune memory with increased chromatin accessibility.

Full text

Before you begin

Innate immune memory refers to the memory status that is ready for responding to the encountering specific or irrelevant secondary stimuli,2 and this state is primarily characterized by epigenetic reprogramming.3 In this protocol, we take advantage of assay for transposase accessible chromatin using sequencing (ATAC-seq) to examine chromatin accessibility and to characterize innate immune memory with enhanced openness.4 Whole glucan particles (WGP) are a well-established inducer of trained immunity1^,^5 and serve as a positive control here for ATAC-seq assay. We conducted all the assays using mouse bone marrow-derived macrophages (BMDMs) and the experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University.

Institutional permissions

The procedures of the experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University.Figure 1. Gating strategy of in vitro differentiated BMDMs (Zombie^−^ F4/80^+^ CD11b^+^)

Culturing of L929 cells and supernatant collection

Timing: about 10 days

• 1.Supplement the complete DMEM medium with GlutaMax to create DMEM-G medium.
• 2.Thaw the cryopreserved L929 cells quickly in a warming bath at 37°C.
• 3.Transfer the cells to a 15 mL tube and add complete DMEM medium to a total volume of 5 mL.
• 4.Centrifuge the tube at 300 × g for 3 min at 25°C.
• 5.Remove the supernatants and resuspend the cell pellets with complete DMEM medium, then transfer them to a T25 flask with a total volume of 5 mL medium.
• 6.Culture the cells in a 37°C incubator with 5% CO_2_ for 3 days. Replace the medium every 3 days until the cells are fully confluent.
• 7.Digest the cells with 0.25% Trypsin-EDTA (1 mL for a T25 flask) in a 37°C incubator for 1 min, then quench the digestion with 4 mL of complete DMEM.
• 8.Transfer the cells to a 15 mL tube and centrifuge cells as in Steps 3 and 4, and resuspend the cell pellets with 1 mL of complete DMEM.
• 9.Count the cells using the TC20 Automated Cell Counter (Bio-Rad) following the manufacturer’s instruction.
• 10.Adjust 1 × 10^5^ cells to a T75 flask with total volume of 15 ml of DMEM-G medium and cultured for 7 days in a 37°C incubator with 5% CO_2_.
• 11.Collect and filter the supernatants using a 0.45 μm syringe filter to obtain L929 conditional medium which contains M-CSF.

CRITICAL: To obtain L929 conditional medium with sufficient M-CSF, the cells are cultured for 7 days without replacing or adding fresh medium. Supernatants must be filtered for collection to avoid contamination by L929 cells. Note: The supernatants can be used immediately or stored at −80°C for up to 6 months, but avoid freeze-thaw cycle.

Isolation and differentiation of BMDMs in vitro

Timing: about 6 days

• 12.Prepare the culture medium for BMDMs (referred to as BMDM-CM), ACK (ammonium-chloride-potassium) lysis buffer for erythrocytes removal and FACS (fluorescence activated cell sorting) staining buffer.
• 13.Euthanize C57BL/6J (B6) mice using CO_2_ and subsequently collect femur and tibia from hinder legs with tissues and skins peeled off.
• 14.Submerge the bare femur and tibia in 75% alcohol for 1–2 min and rinse with 1 × PBS twice.
• 15.Cut the femur and tibia into two fragments at the site of arthrosis.
• 16.Use a 1 mL syringe equipped with a 25 G needle to flush out cells from marrow with 1 × PBS into 100 mm cell culture dishes. Repeat this process with each side for 5 times.
• 17.Disperse the cell cluster by pipetting up and down using a 1 mL pipette for 10 times.
• 18.Filter the cells into a 50 mL tube with a 70 μm cell strainer, and then centrifuge the tube with 300 × g for 5 min at 25°C.
• 19.Discard the supernatants and resuspend the cells with 1 mL of ACK lysis buffer for 1 min.
• 20.Add 5 mL of PBS to quench the lysis, followed by centrifugation (300 × g, 5 min, 25°C) again.
• 21.Resuspend the cell pellets in 25 mL of BMDM-CM, and culture the cells in two 6-well plates at a 37°C incubator with 5% CO_2_ for differentiation.
• 22.Add an additional 2 mL of BMDM-CM on the third day and collect the differentiated BMDMs on the fifth day.
• 23.Digest the BMDMs with 0.25% Trypsin-EDTA (300 μL per well) in a 37°C incubator for 5 min, then quench the digestion with 1 mL of BMDM-CM, followed by centrifugation (300 × g, 5 min, 25°C).
• 24.Resuspend the cell pellets with 1 mL FACS staining buffer and filter the cells through a 100 μm cell strainer to gain single cell suspension.
• 25.Block the Fc receptor (FcR) with mouse anti-CD16/32 antibody (5 μg/mL) at 25°C for 10 min and then wash the cells with FACS staining buffer twice by centrifugation (300 × g, 5 min, 25°C).
• 26.Stain the cells with Zombie NIR dye and two fluorescence-labeled antibodies against F4/80 (FITC, 5 μg/mL) and CD11b (PE, 5 μg/mL) in FACS staining buffer at 4°C for 30 min in the dark.
• 27.Wash the cells twice as pre Step 23, then resuspend the cells in FACS staining buffer and subject to flow cytometry analysis using BD LSR Fortessa X-20 Cell Analyzer.
• 28.Analyze the fluorescence data using the FlowJo software X10.0.7r2 (Tree Star), with an example showing that the differentiated BMDMs constitute over 97% of total cells (Figure 1).

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesAnti-mouse CD11b-PE (M1/70; use 5 μg/mL)BioLegendCat#101208; RRID:AB_312791Anti-mouse F4/80-FITC (BM8; use 5 μg/mL)BioLegendCat#123108; RRID:AB_893502Anti-mouse CD16/32 (93; use 5 μg/mL)BioLegendCat#101301; RRID:AB_312800Chemicals, peptides, and recombinant proteinsDMEM mediumGibcoCat#11965092RPMI 1640 mediumGibcoCat#11875093L929 conditional mediumthis protocolN/AAmmonium chloride (NH_4_Cl)Sigma-AldrichCat#213330Potassium bicarbonate (KHCO_3_)Sigma-AldrichCat#237205Sodium chloride (NaCl)Sigma-AldrichCat#S9888EDTA (0.5 M)Sangon BiotechCat#B5406251 × PBSGibcoCat#10010023Fetal bovine serum (FBS)GibcoCat#102701060.25% Trypsin-EDTAGibcoCat#25200072Penicillin-StreptomycinGibcoCat#15070063β-mercaptoethanolGibcoCat#21985023Sodium pyruvateGibcoCat#11360070GlutaMAXGibcoCat#35050061Antibiotic-AntimycoticGibcoCat#15240062Lipopolysaccharides (LPS)Sigma-AldrichCat#L2630Pam3CSK4InvivoGenCat#tlrl-pmsIFN-γPeproTechCat#315-05HexaneFisher ScientificCat#60-010-54IsopropanolFisher ScientificCat#A461-500MethanolFisher ScientificCat#A452-4Formic acidFisher ScientificCat#A117-2AMPEthanolSangon BiotechCat#A50073724(S),25-epoxycholesterolAbcamCat#ab141633WGP dispersibleInvivoGenCat#tlrl-wgpTRIzolInvitrogenCat#15596026Novoprotein Tagment DNA Extract BeadsNovoproteinCat#N245Novoprotein DNA Clean BeadsNovoproteinCat#N240Critical commercial assaysTNF-α Mouse Uncoated ELISA KitInvitrogenCat#88-7324-88IFN-γ Mouse Uncoated ELISA KitInvitrogenCat#88-7314-88Mouse IFN-β bioluminescent ELISA kit 2.0InvivoGenCat#luex-mifnbv2Chromatin Profile Kit for IlluminaNovoproteinCat#N248Novoprotein NovoNGS Index kit for IlluminaNovoproteinCat#N239Zombie NIR Fixable Viability KitBioLegendCat#423105Cell Counting Kit-8GOONIEBIOCat#100-120Experimental models: Cell linesL929 cell linea gift from Prof. Jun Chen (Sun Yat-sen University Zhongshan School of Medicine)N/AExperimental models: Organisms/strainsMouse: C57BL/6J (6–8 weeks; female or male)Vital River LaboratoryN/ASoftware and algorithmsGraphPad Prism version 9.5.1GraphPad Softwarehttps://www.graphpad.com/featuresFlowJo software X10.0.7r2Tree Starhttps://www.flowjo.com/flowjo/downloadMassHunter Quantitative Analysis SoftwareAgilenthttps://www.agilent.com/en/product/software-informatics/mass-spectrometry-software/data-analysis/quantitative-analysisGeneric Diagramming Platform6BioGDPhttps://biogdp.com/

Materials and equipment

DMEM-GReagentFinal concentrationAmountDMEM mediumN/A444.5 mLFBS10%50 mLPenicillin-Streptomycin1%5 mLGlutaMax2 mM0.5 mLTotalN/A500 mLStore at 4°C for up to 4 weeks. BMDM-CMReagentFinal concentrationAmountRPMI 1640 mediumN/A388.5 mLFBS10%50 mLPenicillin-Streptomycin1%5 mLL929 conditional medium10%50 mLAntibiotic-Antimycotic1%5 mLβ-Mercaptoethanol55 μM0.5 mLSodium Pyruvate1 mM0.5 mLGlutaMax2 mM0.5 mLTotalN/A500 mLStore at 4°C for up to 4 weeks. Alternative: The final concentration of L929 conditional medium can be adjusted from 10 to 20%. Additionally, L929 conditional medium can be substituted with M-CSF at a final concentration of 20–40 ng/mL. ACK lysis bufferReagentFinal concentrationAmountNH_4_Cl150 mM8.02 gKHCO_3_10 mM1 gEDTA (0.5 M)0.1 mM200 μLTotalN/A1 LStore at 25°C–30°C for 1 year. Note: Sterilize using a 0.22 μm syringe filter before use. Alternative: The powder of EDTA or Na_2_EDTA can also be dissolved for ACK, but the final pH should be adjusted between 7.2 and 7.4. FACS staining bufferReagentFinal concentrationAmount1 × PBS (sterilized)N/A48.8 mLFBS2%1 mLEDTA (0.5 M)2 mM200 μLTotalN/A50 mLStore at 4°C for up to 2 weeks. Alternative: 0.5% BSA can serve as alternative for 2% FBS. ATAC lysis bufferReagentFinal concentrationAmount25 × Lysis buffer1 ×2 μL10% NP-400.1%0.5 μL10% Tween-200.1%0.5 μL1% Digitonin0.01%0.5 μLSterilized ddH_2_ON/A46.5 μLTotalN/A50 μLKeep on ice after fresh reconstitution and use immediately. ATAC washing bufferReagentFinal concentrationAmount25 × lysis buffer1 ×40 μL10% NP-400.1%10 μLSterilized ddH_2_ON/A950 μLTotalN/A1 mLKeep on ice after fresh reconstitution and use immediately. ATAC tagmentation bufferReagentFinal concentrationAmount3 × PBS0.3 ×5 μL10% Tween 200.1%0.5 μL1% Digitonin0.01%0.5 μLSterilized ddH_2_ON/A17 μL2 × TD buffer1 ×25 μLTn5 Transposome MixN/A2 μLTotalN/A50 μLKeep on ice after fresh reconstitution and use immediately. Note: The above three buffers included in the Chromatin Profile Kit for Illumina (Cat#N248, Novoprotein) should be freshly reconstituted and gently mixed with a pipette to avoid foam formation. Alternative: Buffers vary among different commercial kits. Furthermore, Hyperactive ATAC-Seq Library Prep Kit for Illumina (Cat#TD711-01, Vazyme) and ATAC-Seq Kit (Cat#53150, Active Motif) are also recommended for use in ATAC-seq assays.

Step-by-step method details

Sample preparation for measuring endogenous 24(S),25-epoxycholesterol

Timing: about 3 days

This step outlines the procedure for preparing samples for mass spectrometric (MS) measurement. Consequently, the hexane and isopropanol used should be of LC-MS grade, and the 2 mL EP tube should be of low binding quality. Prior to cell harvesting, ensure that the hexane and isopropanol are pre-cooled to −20°C and kept over 8 h.

• 1.Digest the mature BMDMs following the previous steps and count the cells using the TC20 Automated Cell Counter (Bio-Rad) according to the manufacturer’s instruction.
• 2.Adjust 1 × 10^7^ cells to a 100 mm dish and culture the cells in a 37°C incubator with 5% CO_2_. Each group requires at least 5 replicates.
• 3.After resting for 24 h, the BMDMs are either harvested directly or treated for the indicated durations.
• 4.Mix the pre-cooled hexane (0.6 volume) and isopropanol (0.4 volume) thoroughly and place the mixture on ice prior to use.
• 5.Remove the medium from the dish and wash the BMDMs twice with pre-chilled 0.9% saline. Note: Sodium chloride is dissolved in ultra-pure H_2_O and sterilized using a 0.22 μm syringe filter. Store at 4°C prior to use.
• 6.Add 1 mL of the hexane/isopropanol mixture to the dish and quickly scrape the cells with a cell scraper on ice, then transfer the contents to a new 2 mL EP tube. CRITICAL: Since the organic mixture is prone to volatilization, this process must be conducted swiftly, with a duration of less than 30 s being ideal. To prevent operational errors, the routes and times for scraping each dish should be kept similar.
• 7.Vortex the scraped mixture at the highest speed for 3 min and chill the EP tube on ice for 2 min.
• 8.Centrifuge the samples at 15,000 × g for 10 min at 4°C.
• 9.Delicately draw the organic supernatants into a new EP tube and then place it on ice. Note: After centrifugation, the mixture separates, carefully transfer the supernatants to a new tube. To avoid contamination by sediments, it is advised to leave approximately 50 μL of supernatant residue.
• 10.Open the tube and dry the mixture thoroughly by blowing nitrogen gas using Reacti-Vap Evaporators (Thermo Scientific). Note: The dried sample is subjected to following measurement immediately or can be stored at −80°C for up to one week.

Sample measurement of 24(S),25-epoxycholesterol

Timing: about 1 day

This method is customized for measuring 24(S),25-epoxycholesterol (24(S),25-EC) in the organic mixture. For other lipid metabolites, the procedures and conditions should be explored case by case accordingly.

• 11.Reconstitute the dried samples with 100 μL of LC-MS grade methanol and vortex thoroughly.
• 12.Utilize the Agilent 6495A Triple Quadrupole mass spectrometer, coupled with an Agilent 1290 Infinity II high-performance liquid chromatography (HPLC) system with an Agilent C18 column (2.1 × 50 mm, 3.0 μm), for the measurement of 24(S),25-EC.
• a.Upload 1 μL of the reconstituted sample or gradient-diluted standard of commercial 24(S),25-EC (Figure 2A) into an Agilent C18 column and keep the temperature of column at 30°C.
• b.The gradient program and flow rate are shown below:Time (min)Flow rate (mL/min)Percentage in phase APercentage in phase B0.000.40158510.000.40010012.900.40010013.000.401585Post-time0.402 minNote: Phase A comprises 90% (v/v) ultra-pure water and 10% methanol (including 0.05% formic acid), while phase B is composed of methanol with 0.05% formic acid.
• c.Quantify the samples using multi reaction monitoring (MRM) mode in positive mode via atmospheric pressure chemical ionization (APCI).Note: The MS parameters are set as follows: a capillary voltage of 3,000 V, a gas temperature of 250°C, a gas flow rate of 11 L/min, a vaporizer temperature of 450°C, and a nebulizer pressure of 35 psi.
• d.Acquire the peak area by collecting ions with a precursor ion set to 383.3 m/z, a product ion of 95 m/z, and a collision energy of 40 eV.
• e.Process the data using Agilent MassHunter Quantitative Analysis software and calculate the concentration by peak area, based on the standard curve from serially diluted standards (Figure 2).CRITICAL: To obtain reliable results, the value of the peak area should fall within the range of the standard curve, thus warranting an appropriate pre-test dilution for the samples.Alternative: A negative control, such as samples pre-treated by inhibitors of enzymes involved in synthesizing the metabolites to be measured, is suggested for the assay. Figure 2. Measurement of endogenous 24(S),25-EC in BMDMs(A) The peak area of gradient-diluted standards and samples.(B) The standard curve for the measured 24(S),25-EC standards.(C) Endogenous level of 24(S),25-EC in BMDMs. Mean ± SEM with five independent samples.Figure 3. Measurement of intracellular 24(S),25-EC with addition of exogenous 24(S),25-EC(A) The measured values of the CCK-8 assay in BMDMs treated with various concentrations of 24(S),25-EC for 24 h.(B) The normalized OD450 of the CCK-8 reaction assay in BMDMs. Mean ± SEM with five independent samples, by one-way ANOVA test followed by Tukey’s multiple comparisons test. n.s., not significant.(C) The peak area of samples with or without the addition of 24(S),25-EC.(D) The intracellular level of 24(S),25-EC in BMDMs. Mean ± SEM with five independent samples, by unpaired t test. ∗∗∗∗p < 0.0001.

Establishing an in vitro-trained immunity model using 24(S),25-epoxycholesterol

Timing: about 8 days CRITICAL: To investigate the role of endogenous metabolites in inducing innate immune memory, it is essential to measure whether these metabolites can enter cells when directly added to medium. If not, permeable derivatives of the metabolites are recommended for further exploration.

• 13.Add 24(S),25-EC (1 μM) to the BMDMs in culture medium directly and incubate for 24 h.

CRITICAL: Before conducting the assay, it is recommended to perform CCK-8 assay to evaluate the impact of metabolites on cellular activity and verify that the concentration is optimal. Besides, freshly generated BMDMs are used to ensure the reliability of the assay. Note: Prepare fresh BMDMs at an appropriate density the day before this assay. For measuring 24(S),25-EC, a 10 cm dish seeded with 1 × 10^7^ cells is recommended. For trained immunity assay, a 24-well culture plate with 5 × 10^5^ cells per well is suggested.

• 14.Harvest the cells from 10 cm dishes and subject them for 24(S),25-EC measurement using the aforementioned procedures (Figure 3).
• 15.Wash the cells in the 24-well plate twice with pre-warmed 1 × PBS.
• 16.Culture the washed cells for another 7 days (resting period) with BMDM-CM at a 37°C incubator with 5% CO_2._ Replace half of the medium with fresh BMDM-CM every 3 days.
• 17.After resting, wash the cells once with 1 × PBS and add fresh medium containing LPS (100 ng/mL).

Alternative: Non-specific stimuli are often used for secondary stimulation. LPS, a TLR4 agonist or Pam3CSK4, a TLR2 agonist, is commonly used. IFN-γ priming prior to restimulation is selective7 and a rest period of 5–7 days is acceptable.

• 18.Collect the culture supernatants after 24 h for ELISA assays immediately or freeze the samples at −20°C.

Note: For RNA isolation, the cells are harvested with TRIzol reagent at 4–6 h post restimulation. The RNA is used for either qPCR analysis or transcriptomic sequencing.

ELISA assay measuring cytokine concentration

Timing: about 2 days

The ELISA tests are performed in accordance with the guidelines provided by the manufacturer for the commercial kits listed in the key resources table.

• 19.Coat 100 μL of diluted capture antibodies into high-binding 96-well plates with 1 × coating buffer and incubate over 16 h at 4°C. Alternative: 1 × coating buffer can be substituted with 1 × PBS (pH 7.4) buffer. Both buffers should be filtered through a 0.22 μm syringe filter.CRITICAL: Coating buffer must be free of any carrier proteins, including BSA and FBS. Besides, high-binding plates are crucial for the effective binding of antibodies.
• 20.Wash the plates 3 times with 200 μL washing buffer per well and tap the plates on absorbent paper towels to remove any residual buffer. Note: The washing buffer is composed of 1 × PBS with 0.05% (v/v) Tween-20. After taping, subsequent operations should be performed swiftly to keep the plate from drying out for too long.
• 21.Block the plates with 200 μL of 1 × ELISA/ELISPOT buffer and incubate for at least 1 h on a shaker at 25°C–30°C. Alternative: 1 × ELISA/ELISPOT buffer can be substituted with 1 × PBS containing 1% BSA or heat-inactivated FBS.
• 22.Prepare the standards and dilute the samples.
• a.Reconstitute and dilute the standards in serial dilutions.
• b.Dilute the samples with 1 × ELISA/ELISPOT buffer or PBS in an appropriate dilution fold.
• 23.Repeat Step 20 and then incubate 100 μL of diluted samples or standards in individual wells of the plates for at least 2 h on a shaker at 25°C–30°C. Note: The dilution fold and incubation time for different samples can be optimized. For detecting IFN-γ, over 16 h incubation of the samples at 4°C is recommended.
• 24.Repeat Step 20 and add 100 μL of diluted detection antibody to each well, then incubate on a shaker for 1 h at 25°C–30°C.
• 25.Repeat Step 20 and add 100 μL of diluted avidin-HRP or streptavidin-HRP (horseradish peroxidase) into each well, then incubate in the dark for 30 min at 25°C–30°C.
• 26.Wash the wells with 200 μL of washing buffer 5 times and tap the plates on absorbent paper towels to remove any residual buffer.
• 27.Add 100 μL of HRP substrate (TMB; 3,3,5,5-tetramethylbenzidine) and incubate in the dark for 10–20 min. CRITICAL: The incubation of both TMB and HRP should occur in the dark. TMB reaction is monitored in real-time and quenched at appropriate time based on the color intensity of the samples.
• 28.Add 50 μL of stop solution into the wells to terminate the reaction. Note: The stop solution is composed of 2 M H_2_SO_4_ or 2 M HCl and stored at 25°C–30°C.
• 29.Measure the absorbance at 450 nm using a microplate reader and set the absorbance at 570 nm as reference.
• 30.Calculate the concentration of the samples in accordance with the standards (Figure 4). CRITICAL: The dilution fold should be optimized to ensure that the absorbance values of the samples fall within the range of the standards.Figure 4ELISA assay measuring TNF-α, IFN-β, and IFN-γ in the supernatants from 24(S),25-EC-trained BMDMs(A) The standard curve of TNF-α, IFN-β and IFN-γ standards.(B) The supernatant levels of TNF-α, IFN-β and IFN-γ in trained BMDMs after LPS restimulation. Mean ± SEM with three independent experiments, by two-way ANOVA test followed by Sidak’s multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.001.

ATAC-seq

Timing: 30 min (for step 31) Timing: 30 min (for step 32) Timing: 50 min (for step 33) Timing: 30 min (for step 34) Timing: 60 min (for step 35) Timing: 30 min (for step 36)

The detection of cytokines can reflect the enhanced secondary inflammatory response after training,8 but to define the innate immune memory after initial training, the epigenetic changes such as chromatin openness need to tested.9 For this purpose, ATAC-seq is performed to investigate chromatin accessibility and to reflect the induction of immune memory. Thus, we extract the nuclei from 24(S),25-EC-trained BMDMs on the last day of the resting period to construct an ATAC-seq library.

• 31.Collect live BMDMs.
• a.Wash the cells with warm 1 × PBS once, then incubate the cells with Trypsin-EDTA (0.25%) for 1 min at 37°C, and wash the cells again with 1 × PBS twice.
• b.Digest BMDMs with Trypsin-EDTA (0.25%) for 5 min at 37°C.
• c.Stop the digestion with BMDM-CM and collect the cells through centrifugation at 150 × g for 3 min at 25°C.
• d.Count the cells and adjust the cell concentration to 1 × 10^5^ cells in 100 μL of 1 × PBS.CRITICAL: The cells prepared for the assay must contain over 95% live cells. This is crucial because the nucleosomes in dead cells dissociate, which can potentially skew the actual state of chromatin accessibility and reduce the signal-to-noise ratio (S/N). To exclude dead cells, pre-incubate the cells with Trypsin-EDTA (0.25%) for a short duration can remove the low-adherent dead cells, then centrifuge at a low speed to retain only the live cells. Lastly, we stained the cells with trypan blue10 to count and assess the ratio of live cells (above 95%) using the TC20 Automated Cell Counter (Bio-Rad) according to the manufacturer’s instruction.
• e.Centrifuge the cells at 500 × g for 5 min at 25°C, resuspend with 100 μL of 1 × PBS, and centrifuge again at 500 × g for 5 min.
• f.Remove the supernatants carefully and place the semi-dry cells on ice, ready for use.
• 32.Isolate the nuclei from live BMDMs.
• a.Prepare the ATAC lysis buffer and ATAC washing buffer, and place them on ice for use.
• b.Add 45 μL of pre-cooled ATAC lysis buffer to the tubes and gently pipette the cells up and down for 10 times. Resuspend and lyse the cells on ice for 5 min.
• c.Gently pipette the lysed cells with 950 μL of ATAC washing buffer up and down for 5 times.
• d.Centrifuge the cells at 500 × g for 5 min at 4°C.
• e.Carefully place the tube on ice and open the lids to remove the supernatant. Note: To prevent the loss of nuclei, initially use a 1 mL pipette to leave about 50 μL supernatant. Subsequently, use a 20 μL pipette to remove the remaining supernatant, allowing up to 5 μL supernatant uncollected.
• 33.Chromatin tagmentation.
• a.Prepare the ATAC tagmentation buffer and place it on ice for use.
• b.Resuspend the nuclei with 40 μL of chromatin tagmentation buffer by pipetting up and down for 10 times, and then transfer the suspension into a new 200 μL PCR tube.
• c.Incubation the tube at 37°C for 30 min in a PCR Amplifier.
• d.Add 5 μL of stop buffer into the PCR tube, vortex thoroughly and incubation again at 55°C for 10 min after a brief centrifugation. Note: The stop buffer is composed of 5% SDS, and the samples post-reaction can be subjected to purification or stored at −20°C for up to 1 week.
• 34.Purification of the tagmentated chromatins.
• a.Pre-warm the Tagment DNA Extract Beads in 25°C for at least 15 min.
• b.Vortex the beads thoroughly for 30 s and add 2 volume beads (i.e., 90 μL) into the PCR tube. Mix the beads by pipetting up and down, and incubate at 25°C for 5 min.
• c.Briefly centrifuge the tube and place it in a magnetic hopper grate at 25°C–30°C for 5 min.
• d.Carefully remove the liquid and wash the beads with 200 μL of fresh 80% ethanol twice (1 min for each washing).CRITICAL: Always keep the tube in the magnetic hopper grate during washing and avoid the beads from being flushed directly.Alternative: If a 1.5 mL tube is used for purification, 1 mL of 80% ethanol is employed for washing each time.
• e.Thoroughly remove the 80% ethanol and dry the beads in the magnetic hopper grate for about 4–6 min.Note: The drying time for the beads varies depending on the environmental temperature, so check the beads in real-time. If the edges of beads turn yellow and the surface become dull, the beads can be considered dry.
• f.Take the tube away from the magnetic hopper grate and add 22 μL of elution buffer to resuspend the beads. Incubation at 25°C for 3 min.Note: If the beads naturally settle to the bottom, use the pipette to resuspend again and incubate for extra 2 min.
• g.Briefly centrifuge the tube and place the tube in the magnetic hopper grate again for 2 min.
• h.Transfer the supernatants (about 21 μL) to a new tube. The purified DNAs can be subjected to amplification or stored at −20°C for up to 1 month.
• 35.Library amplification.
• a.Prepare the Library amplification mix in a new 200 μL PCR tube.ReagentAmountPurified DNA16 μLi5 index2.5 μLi7 index2.5 μL2 × HiFi AmpliMix25 μLSterilized ddH_2_O4 μLTotal50 μL
• b.Vortex the mix and subject it to a PCR Amplifier after a brief centrifugation and subsequent amplification procedure.StepsTemperature (°C)Time (s)CyclesStrand Displacement721801Initial Denaturation98301Denaturation981513Annealing638Extension723Final extension721201Hold10∞1CRITICAL: The amplification cycles vary depending on cell type and cell number. To explore the appropriate cycles, 1 μL purified DNA is used to amplify with three gradient cycles (n) and followed by agarose gel electrophoresis (AGE). The cycles (n-4) were determined according to results of AGE, as the detectable DNAs are sufficient for sequencing (Figure 5). Since there may be single-stranded gaps between the adapters and the inserted fragments, a strand displacement reaction at 72°C for 3 min is required to fill these gaps.
• 36.Purification of the library. Figure 5ATAC-seq assay measuring the chromatin accessibility of 24(S),25-EC-trained BMDMs(A) AGE of amplified DNA libraries with different amplifying cycles.(B) Density plots and heatmaps of all peaks depict the global chromatin accessibility around TSS regions in each group. Kb stands for kilobase and TSS stands for transcription start site. WGP serves as a positive control for inducing innate immune memory with increased chromatin accessibility.

The procedures for purifying library are similar to Step 34 except for that the DNA Clean Beads are used and the volume is 1.3 fold. The purified library is ready for quality control (QC), sequencing, and analysis (Figure 5).Alternative: The binding of the beads can be divided into two steps to enhance the purity of library. First, bind the PCR products with 0.55 volume of beads, then transfer the supernatants to a new tube containing 0.75 volume of fresh beads. After undergoing secondary binding, the beads are subjected to washing with 80% ethanol.

Expected outcomes

This protocol aims to establish a method for measuring endogenous metabolites, especially the lipid metabolites, and to examine their impact on inducing innate immune memory. Therefore, the protocol would measure the intracellular level of 24(S),25-EC in mouse macrophages (as shown in Figure 2). Besides, the protocol would also indicate that 24(S),25-EC is cell-permeable (as shown in Figure 3). Moreover, the protocol would show that 24(S),25-EC induces innate immune memory in treated BMDMs, exhibiting generally higher chromatin accessibility (refer to Figure 5B), and displays trained immunity features in trained BMDMs, evidenced by enhanced TNF and IFNs production upon restimulation with LPS (refer to Figure 4).

Quantification and statistical analysis

The fluorescence data is analyzed using FlowJo software version X10.0.7r2 (Tree Star), and the mass spectrometry data is processed with Agilent MassHunter Quantitative Analysis software (Agilent). The GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, California, USA) is used for further data analysis. Results are presented as the mean ± SEM. Statistical analysis is performed using an unpaired t-test, one-way or two-way ANOVA, followed by Tukey’s multiple comparisons test, as appropriate. All statistical significance and comparisons are detailed in the figure legends.

Limitations

The in vitro model for inducing innate immune memory may not sufficiently reflect the complexity of in vivo physiological conditions. This protocol is adapted for mouse BMDMs and needs further optimization for other types of cells or species. The addition of 24(S),25-EC can induce innate immune memory in BMDMs; however, it remains unknown whether 24(S),25-EC secreted by cells can enter and affect BMDMs. Although our previous literature has indicated that 24(S),25-EC induces trained immunity by activating endogenous receptors,1 this protocol could not determine whether surface receptors were also necessary, similar to mevalonate11 and oxidized low-density lipoprotein (oxLDL).12

Troubleshooting

Problem 1

The quantity and cell status of differentiated BMDMs are not adequate for subsequent assays.

Potential solution

It may be due to insufficient levels of the M-CSF for macrophage differentiation; thus it is suggested to increase the proportion of L929 conditional medium or the concentration of M-CSF. Additionally, the ethanol must be thoroughly washed off.13

Problem 2

There are very large variations in the concentration of 24(S),25-EC among the samples in the group.

Potential solution

To ensure consistency, the cells in replicates should initially be kept in equal amounts. Furthermore, the method of scraping cells should be kept as similar as possible. Additionally, the reagents used for extracting metabolites must be of LC-MS grade to prevent any unnecessary or unspecific contamination.

Problem 3

The level of cytokines following secondary stimulation in trained BMDMs does not increase compared to that of untreated BMDMs.

Potential solution

The issue may stem from two potential reasons. One is that 24(S),25-EC is inactive, and the other is that the cells may be contaminated prior to the first stimulation. Therefore, 24(S),25-EC must be divided into aliquots and repeated freeze-thaw cycles should be avoided. Additionally, it is essential to ensure that the mice used are healthy, and that the cells are free from mycoplasma or fungal contamination during the operation.

Problem 4

The concentration of the library is too low for sequencing.

Potential solution

The low concentration of the library is primarily due to the loss of nuclei. When extracting nuclei, the pipette should be used to avoid contact with the cells during the removal of supernatants and should be handled with care. Besides, increasing the original cell numbers will improve the concentration of the library. Furthermore, always optimize the amplification cycles to ensure the yield of the library.

Problem 5

Band dispersion in the testing agarose gel is irregular when amplifying the library.

Potential solution

It may be attributed to two possible reasons. One is the low activity of the cells, and the other is the contamination of mycoplasma. The DNA of the dead cells or mycoplasma is more exposed than that in clean and live cells, so the chromatins are randomly digested by Transposase without the protection of nucleosomes and become more dispersed. To address this, the cells should be kept clean and dead cells should be removed as thoroughly as possible before nuclei extraction.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Jinyun Liu (hy0224009@muhn.edu.cn).

Technical contact

Details and questions for this protocol should be directed to and answered by Yongxiang Liu (liu_yongxiang@gzlab.ac.cn).

Materials availability

This protocol did not produce any new materials.

Data and code availability

This protocol did not generate any new code, and all data were included in the study.

Acknowledgments

This work was supported by grants from the 10.13039/100014717National Natural Science Foundation of China (32370923 and 32400716) and Young Scientists Program of Guangzhou National Laboratory (QNPG24-03). The graphical abstract was created from BioGDP.com with partial modifications.

Author contributions

Protocol design, Y.L., J.L., and X.X.; performing assays, Y.L., H.J., and B.H.; writing, Y.L.; revising, Y.L., X.X., and J.L.

Declaration of interests

The authors declare no competing interests.