Protocol for measuring fatty acid absorption and intracellular lipid accumulation in intestinal cells and organoids

Summary

Quantitative assessment of intestinal epithelial cells is essential for studies of metabolism and disease. Here, we present a protocol to quantify fatty acid uptake and lipid accumulation in intestinal epithelial cell lines and primary intestinal organoids using a flow cytometry-based approach with BODIPY FL C₁₆. We describe steps for assessing fatty acid uptake and accumulation in intestinal epithelial cell lines and intestinal organoids. This rapid and reproducible technique can also be adapted to investigate fatty acid metabolism in other cell types.

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

Graphical abstract

Highlights

• •Isolate and culture mouse small intestinal crypt-derived organoids
• •Label intestinal cells and organoids with BODIPY FL C₁₆ for fatty acid uptake
• •Quantify fatty acid uptake by flow cytometry immediately after labeling
• •Measure intracellular lipid accumulation following a defined chase period

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

Quantitative assessment of intestinal epithelial cells is essential for studies of metabolism and disease. Here, we present a protocol to quantify fatty acid uptake and lipid accumulation in intestinal epithelial cell lines and primary intestinal organoids using a flow cytometry-based approach with BODIPY FL C₁₆. We describe steps for assessing fatty acid uptake and accumulation in intestinal epithelial cell lines and intestinal organoids. This rapid and reproducible technique can also be adapted to investigate fatty acid metabolism in other cell types.

Before you begin

The intestinal epithelium is the primary site of dietary lipid absorption, where fatty acids are taken up by enterocytes, re-esterified into triglycerides, and packaged into chylomicrons for transport to peripheral tissues. Tight regulation of these processes is essential for systemic energy homeostasis, whereas impaired lipid handling can result in intracellular lipid accumulation, lipotoxicity, inflammation, and metabolic disease. Quantitative assessment of fatty acid uptake and lipid accumulation is therefore critical for investigating intestinal lipid metabolism under physiological and pathological conditions.

Conventional approaches for measuring fatty acid uptake, including radiolabeled tracers and fluorescence microscopy, have significant limitations. Radiotracer-based assays require cell lysis and provide only population-level measurements, whereas microscopy offers spatial information but is relatively low throughput and less suitable for quantitative analysis of large sample numbers or organoid-derived cells.

Here, we describe a flow cytometry-based protocol using the fluorescent long-chain fatty acid analog BODIPY FL C₁₆ to quantify fatty acid uptake and lipid accumulation in intestinal epithelial cell lines and dissociated intestinal organoids. The assay enables rapid, quantitative analysis at single-cell resolution while preserving cellular integrity and is readily scalable for high-throughput studies. Because the workflow is simple and requires only standard flow cytometry instrumentation, it can be broadly applied to investigate fatty acid metabolism in diverse cell types capable of lipid uptake.

Innovation

This protocol adapts BODIPY FL C₁₆ labeling for flow cytometric quantification of cellular fatty acid uptake and lipid accumulation, providing a rapid, quantitative, and high-throughput alternative to confocal microscopy. Unlike confocal imaging, which is limited by manual field selection, small sample sizes, and low throughput, flow cytometry enables objective analysis of thousands of individual cells per sample with minimal sampling bias. The assay uses lower concentrations of BODIPY FL C₁₆ than are typically required for confocal imaging while maintaining robust fluorescence signals, reducing probe consumption and experimental cost. It is readily applicable to both intestinal epithelial cell lines and dissociated intestinal organoids, enabling quantitative analysis in physiologically relevant models. By combining single-cell resolution, scalability, reproducibility, and compatibility with standard flow cytometers, this protocol expands access to high-throughput studies of fatty acid uptake and intracellular lipid accumulation across diverse experimental systems.

Institutional permissions (if applicable)

All animal procedures were approved by the Animal Care and Use Committee of Fudan University and conducted in accordance with institutional guidelines. All animals were housed under specific pathogen-free (SPF) conditions at the Fudan University Animal Center.

Preparation of BODIPY FL C₁₆ stock solution and storage

Timing: 20 min

Here, we describe the essential steps for preparing and storing the BODIPY FL C₁₆ stock solution. Since this fluorescent probe is used to evaluate lipid metabolism capacity in intestinal epithelial cells, its proper handling is critical for assay reproducibility and sensitivity.

• 1.Briefly centrifuge the vial containing BODIPY FL C₁₆ powder to collect all powder at the bottom.

 Place the sealed vial in a benchtop centrifuge and spin at 1,000× g for 10–15 s.

CRITICAL: This step prevents powder loss when opening the vial.

• 2.
  Transfer the vial to a biosafety cabinet and add sterile DMSO to achieve a 2 mM stock solution.

  • a.
    Calculate the required volume of DMSO based on the manufacturer’s specified mass of BODIPY FL C₁₆.
  • b.
    Aseptically add DMSO directly into the vial.

CRITICAL: Ensure the DMSO is sterile and anhydrous to avoid degradation of the fluorescent dye.

• 3.Mix the solution gently to ensure complete dissolution.

 Use a pipette to gently pipette up and down 10–15 times, or slowly invert the sealed vial several times.

• 4.
  Prepare small-volume aliquots of the 2 mM stock solution and store them properly.

  • a.
    Using a sterile pipette, dispense 5 μL aliquots of the stock solution into low-adhesion, sterile microcentrifuge tubes. Each aliquot can be used for up to 2 freeze-thaw cycles (covering 2–3 experiments).
  • b.
    Label the tube with the concentration, date, and number of freeze-thaw cycles.

    CRITICAL: Aliquot the reagent before storage to avoid repeated freeze–thaw cycles, which can reduce fluorescence intensity.

  • c.
    Store the aliquots at −20°C, protected from light.

    • i.
      Place the aliquots in a light-tight container, or wrap the tubes in aluminum foil.
    • ii.
      Under these conditions, the stock solution remains stable for up to one year.

      CRITICAL: Thaw only one aliquot at a time; use it immediately and discard any unused portion. Gently mix the thawed aliquot by pipetting.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
BODIPY™ FL C16	ThermoFisher	Cat#D3821
DMEM	Servicebio	Cat#G4511
DMEM/F12 50:50 (L-glutamine,15mM HEPES)	MACGENE	Cat#CM10092
Fetal Bovine Serum	Vazyme	Cat#F101-01
MEM-Eagle	Viva Cell	Cat#C3240-0100
Penicillin-Streptomycin Nystatin Solution (100∗)	Viva Cell	Cat#C3422-0100
Matrigel	Corning	Cat#356255
EGF	Novoprotein	Cat#C029
R-spondin 1	Novoprotein	Cat#CX83
Noggin	Novoprotein	Cat#C028
Y-27632	MedChemExpress	Cat#HY10071
Propidium Iodide	MedChemExpress	Cat#HYD0815
DMSO	Solarbio	Cat#D8370
TrypLE™ Express	ThermoFisher	Cat#12604013
Recombinant Human IL-22	Novoprotein	Cat#CH50
Lomitapide	Targetmol	Cat#T6267
Trypsin EDTA Solution B	VivaCell	Cat#C3532
Bovine Serum Albumin	Sigma-Aldrich	Cat#SRE0098-50G
Experimental models: Cell lines
Caco-2	ATCC	Cat#HTB-37; RRID:CVCL_0025
Experimental models: Organisms/strains
Mouse: C57BL/6JGpt (no sex restriction; wild type; 5–6 weeks)	GemPharmatech	Cat#N000013
Software and algorithms
GraphPad Prism(version 9.5.1)	GraphPad Software	https://www.graphpad.com/
FlowJo (version 10.8.1)	BD Biosciences	https://flowjo.com/
Other
96-well U-bottom plate	Nantong Uni-Jia Experimental Equipment Co., Ltd.	Cat#HB-PY-96-U
Full spectrum flow cytometer Aurora	Cytek	N/A

Materials and equipment

20 mM EDTA/PBS

Reagent	Final concentration	Amount
EDTA (0.5 M)	20 mM	1.2 mL
PBS (10 ×)	1 ×	3 mL
ddH2O	N/A	25.8 mL
Total	N/A	30 mL

Note: This solution should be prepared fresh and kept on ice after preparation for later use.

Organoid medium

Reagent	Final concentration	Amount
DMEM/F12 50:50 (L-glutamine,15mM HEPES)	N/A	7.87 mL
FBS	20%	2 mL
Penicillin-Streptomycin Nystatin Solution (100 ×)	1%	1.1 mL
EGF (100 μg/mL)	50 ng/mL	5 μL
R-spondin 1 (1 mg/mL)	500 ng/mL	5 μL
Noggin (100 μg/mL)	100 ng/mL	10 μL
Y-27632 (10 mM)	10 μM	10 μL
Total	N/A	10 mL

Note: This solution should be prepared fresh and kept on ice after preparation for later use.

Step-by-step method details

Protocol for assessing lipid uptake in intestinal epithelial cell lines

Timing: 6 h

Here, we describe steps for the quantitative assessment of fatty acid uptake in intestinal epithelial cell lines using a validated flow cytometry-based assay with the fluorescent fatty acid analog BODIPY FL C₁₆.

Note: Accurate quantification of fatty acid uptake in intestinal epithelial cell lines requires stringent control of serum deprivation, starvation conditions, and protection of fluorescent reagents from light. Standardization of these critical steps minimizes experimental variability and ensures reproducible measurements.

• 1.Prepare healthy cells for the assay:

 Use Caco-2 cells in the logarithmic growth phase with viability ≥90% and a confluent, dense monolayer.

Note: If cells are required to stimulate specific cytokines, initiate stimulation at an appropriate time; stimulation may also be maintained during the subsequent starvation period. Adjust the timing based on your experimental conditions.

Note: 1 × 10⁵ cells per well in a 48-well plate are sufficient for this assay; adjust the cell number flexibly based on your cell line's characteristics.

• 2.
  Remove complete medium and wash:

  • a.
    Aspirate the complete medium.
  • b.
    Wash each well 2–3 times with PBS.

CRITICAL: Wash cells thoroughly. Residual serum (FBS) can interfere with assay results, leading to unreliable data.

• 3.
  Starve cells in serum-free medium:

  • a.
    Add serum-free medium to each well.
  • b.
    Incubate for 4 h to starve.
• 4.
  Wash cells after starvation:

  • a.
    Aspirate the serum-free medium.
  • b.
    Wash each well 2–3 times with PBS.
• 5.
  Incubate with BODIPY FL C₁₆:

  • a.
    Dilute BODIPY FL C₁₆ to 0.1 μM in 0.1% BSA/PBS.
  • b.
    Add 200 μL of BODIPY FL C₁₆/BSA solution per well.
  • c.
    For blank controls, use 0.1% BSA/PBS without BODIPY FL C₁₆.
  • d.
    Transfer the plate to the incubator.

CRITICAL: Perform all steps from this point onward in the dark or under minimal light.

• 6.Incubate at 37°C for 20 min.
• 7.
  Stop the incubation and wash:

  • a.
    Add 400 μL ice-cold 0.2% BSA/PBS to each well to stop the incubation.
  • b.
    Quickly aspirate the supernatant.
  • c.
    Wash each well 2–3 times with ice-cold PBS.
• 8.
  Detach cells with trypsin:

  • a.
    Aspirate the residual PBS from the wells.
  • b.
    Add 30–50 μL Trypsin per well.
  • c.
    Incubate at room temperature (25°C) for 2 min.
• 9.
  Terminate digestion and transfer to a U-bottom plate:

  • a.
    Add 150 μL 10% FBS/PBS to stop trypsinization.
  • b.
    Transfer the cell suspension to a 96-well U-bottom plate.
  • c.
    Centrifuge at 1,500 rpm for 5 min at 4°C.
  • d.
    Discard the supernatant.
• 10.Resuspend the single-cell pellet in 2% FBS/PBS.
• 11.
  Stain for viability:

  • a.
    Immediately before the flow cytometry analysis, add PI dye (final concentration 1μg/mL) to label dead cells.

CRITICAL: After BODIPY FL C₁₆ incubation, keep all samples on ice and protected from light, and perform flow cytometry as soon as possible.

• 12.Perform flow cytometry analysis on the prepared single-cell suspension to measure BODIPY FL C₁₆ fluorescence (505/512 nm) and PI staining (535/617 nm).

Protocol for assessing lipid accumulation in intestinal epithelial cell lines

Timing: At least 7 h

Here, we describe steps for a validated flow cytometry-based assay using BODIPY FL C₁₆ to measure fatty acid accumulation in intestinal epithelial cell lines via sequential uptake, washout, and accumulation culture.

Note: Quantification of fatty acid accumulation in intestinal epithelial cell lines is highly dependent on standardized post-uptake culture conditions, including the timing and duration of cytokine stimulation. Careful control of these parameters minimizes experimental variability and ensures reproducible assessment of intracellular lipid retention.

• 13.Prepare healthy Caco-2 cells for the assay:

 Use cells in the logarithmic growth phase with viability ≥90% and a confluent, dense monolayer.

Note: If cytokine stimulation is required, it should generally be performed after BODIPY FL C₁₆ uptake. Cytokines should be added before BODIPY FL C₁₆ incubation only if prior validation has demonstrated that the treatment does not alter fatty acid uptake.

Note: 1 × 10⁵ cells per well in a 48-well plate are sufficient for this assay; adjust the cell number flexibly based on your cell line's characteristics.

• 14.
  Remove complete medium and wash:

  • a.
    Aspirate the complete medium.
  • b.
    Wash each well 2–3 times with PBS.

CRITICAL: Wash cells thoroughly. Residual serum (FBS) can interfere with assay results, leading to unreliable data.

• 15.
  Starve cells in serum-free medium:

  • a.
    Add serum-free medium to each well.
  • b.
    Incubate for 4 h to starve.
• 16.
  Wash cells after starvation:

  • a.
    Aspirate the serum-free medium.
  • b.
    Wash each well 2–3 times with PBS.
• 17.
  Incubate with BODIPY FL C₁₆:

  • a.
    Dilute BODIPY FL C₁₆ to 0.1 μM in 0.1% BSA/PBS.
  • b.
    For blank controls, use 0.1% BSA/PBS without BODIPY FL C₁₆.
  • c.
    Add 200 μL of BODIPY FL C₁₆/BSA solution per well.
  • d.
    Transfer the plate to the incubator.

CRITICAL: Perform all steps from this point onward in the dark or under minimal light.

• 18.Incubate at 37°C for 30 min.
• 19.
  Stop uptake and start accumulation:

  • a.
    Add 400 μL ice-cold 0.2% BSA/PBS to each well to stop the incubation.
  • b.
    Quickly aspirate the supernatant.
  • c.
    Wash each well 2–3 times with ice-cold PBS.
  • d.
    Add complete medium to each well.

Notion: If cytokine stimulation is required, add the cytokine at this step. Cytokines should not be added before BODIPY FL C₁₆ incubation unless prior validation has confirmed that the treatment does not alter fatty acid uptake.

Notion: Culture the cells for the desired duration according to the experimental design.

• 20.
  Detach cells with trypsin:

  • a.
    Aspirate the culture medium.
  • b.
    Wash each well 2–3 times with PBS.
  • c.
    Aspirate the residual PBS from the wells.
  • d.
    Add 30–50 μL Trypsin per well.
  • e.
    Incubate at room temperature (25°C) for 2 min.
• 21.
  Terminate digestion and transfer to a U-bottom plate:

  • a.
    Add 150 μL 10% FBS/PBS to stop trypsinization.
  • b.
    Transfer the cell suspension to a 96-well U-bottom plate.
  • c.
    Centrifuge at 1,500 rpm for 5 min at 4°C.
  • d.
    Discard the supernatant.
• 22.Resuspend the single-cell pellet in 2% FBS/PBS.
• 23.
  Stain for viability:

  • a.
    Immediately before the flow cytometry analysis, add PI dye (final concentration 1 μg/mL) to label dead cells.

CRITICAL: After BODIPY FL C₁₆ incubation, keep all samples on ice and protected from light, and perform flow cytometry as soon as possible.

CRITICAL: Prepare an additional 1–2 wells following the fatty acid uptake protocol described above, with a 30 min BODIPY FL C₁₆ incubation instead of 20 min, omitting cytokine stimulation and the subsequent incubation period, to serve as a fluorescence baseline control.

• 24.Perform flow cytometry analysis on the prepared single-cell suspension to measure BODIPY FL C₁₆ fluorescence (505/512 nm) and PI staining (535/617 nm).

Protocol for assessing lipid uptake in intestinal organoids

Intestinal organoids preserve the cellular diversity and three-dimensional architecture of the native intestinal epithelium provides a physiologically relevant model for studying lipid metabolism. Here, we describe steps for intestinal crypt isolation² and provide a detailed protocol for organoid culture³ for reference.

Intestinal crypt isolation and organoid culture

Timing: 7 days

• 25.
  Isolate duodenum and proximal jejunum segments:

  • a.
    Euthanize the mouse by cervical dislocation and dissect out the small intestine.
  • b.
    Flush the lumen with ice-cold PBS to remove contents.
  • c.
    Cut the desired segment (duodenum and approximately 10 cm of proximal jejunum).
  • d.
    Remove the mesentery and Peyer’s patches, and cut the intestine longitudinally to open it.
  • e.
    Soak the tissue in ice-cold PBS containing Penicillin-Streptomycin Nystatin Solution (1:50 penicillin/streptomycin:PBS).

CRITICAL: Keep the tissue on ice at all steps to preserve viability.

• 26.
  Incubate and remove supernatants:

  • a.
    Repeat the following steps twice: add 10 mL of 20 mM EDTA in PBS, incubate on ice for 30 min, vortex for 30 s at 700 × g (2000 rpm), then discard the supernatant.
  • b.
    Third incubation:

    • i.
      Add 10 mL of 20 mM EDTA/PBS (prepare fresh and use immediately).
    • ii.
      Incubate on ice for 30 min.
    • iii.
      Vortex for 1 min to fully release the crypts.
    • iv.
      Collect the supernatant (which contains isolated crypts).
• 27.
  Filter, collect, and wash crypts:

  • a.
    Prepare 5% FBS/PBS. Rinse a 50 mL centrifuge tube and a 70 μm cell strainer with this solution.
  • b.
    Filter the supernatant containing the crypts through the strainer.
  • c.
    Centrifuge at 300 × g for 5 min at 4°C. Remove the supernatant.
• 28.
  Wash and resuspend crypts:

  • a.
    Wash the crypts with PBS, centrifuge at 300 × g for 2 min at 4°C, and remove the supernatant. Repeat wash twice.
  • b.
    Add ice-cold DMEM/F12 medium, resuspend the pellet to wash away residual mucus and dead cells.
  • c.
    Centrifuge at 200 × g for 5 min at 4°C and remove the supernatant.
  • d.
    Resuspend the crypts in an appropriate volume of small intestinal organoid medium and count them under a microscope.
• 29.
  Seed crypts in Matrigel:

  • a.
    Dilute the crypts to a suitable concentration (microscopic examination of the sample revealed a count of 150 to 200 crypts per 20 μL).
  • b.
    Mix crypts and Matrigel at a 1:1 volume ratio.
  • c.
    Seed 25 μL per well into a 48-well plate (50–100 crypts per well).
  • d.
    Incubate at 37°C for 8–10 min to allow Matrigel to solidify.

CRITICAL: Keep Matrigel and crypts on ice during mixing to prevent premature gelation.

• 30.
  Culture and observe organoids:

  • a.
    Add 300 μL of organoid medium (prepare fresh and use immediately) per well, then transfer the plate to the incubator.
  • b.
    Observe daily and change the medium when it turns yellow (usually on the third and fifth day).
  • c.
    Viable crypts become spherical within about 1 hour after seeding. Budding typically occurs between days 3–5 of culture.

Building on the fatty acid uptake assay and the intestinal organoid culture protocols described above, we developed and describe here a flow cytometry-based approach using BODIPY FL C16 to quantify fatty acid uptake in dissociated intestinal organoids. Here, we provide steps for BODIPY FL C₁₆ labeling and incubation, organoid dissociation, and subsequent flow cytometric acquisition, enabling quantitative analysis of intestinal lipid metabolism in a physiologically relevant model that more closely recapitulates the in vivo environment.

Measurement of lipid uptake in intestinal organoids

Timing: 6 h

• 31.
  Prepare organoids for the absorption assay:

  • a.
    Use well-differentiated organoids cultured for 7–8 days.
  • b.
    If external reagent stimulation is required, start adding the reagent on day 7.

Note: Because reagents must penetrate the Matrigel to reach organoids, if your reagent requires a short stimulation time in cell lines, extend the stimulation time appropriately for organoids. For very short treatments (≤1 h), remove the Matrigel before stimulation, then wash thoroughly before proceeding to the starvation step.

• 32.
  Starve organoids in serum-free medium:

  • a.
    Remove complete medium.
  • b.
    Wash each well 2–3 times with PBS.
  • c.
    Add serum-free DMEM/F12 medium (300 μL per well).
  • d.
    Incubate for 4 h to complete starvation.
• 33.
  Remove Matrigel:

  • a.
    Aspirate the medium from the well.
  • b.
    Add 300 μL ice-cold PBS, leave for 30 s – 1 min.
  • c.
    Gently pipette the gel 1–2 times until it dissolves.
  • d.
    Transfer the suspension to a 1.5 mL EP tube.
  • e.
    Centrifuge at 200 × g for 2 min at 4°C. Discard the supernatant.
  • f.
    Add 1 mL ice-cold PBS, gently pipette to resuspend, then centrifuge again at 200 × g for 2 min at 4°C.
  • g.
    Repeat the wash (step f) twice more.

CRITICAL: Before incubating with BODIPY FL C₁₆, ensure Matrigel is completely removed.

• 34.
  Incubate with BODIPY FL C₁₆:

  • a.
    Dilute BODIPY FL C₁₆ stock solution to 0.5 μM in 0.1% BSA/PBS.
  • b.
    For blank controls, use 0.1% BSA/PBS without BODIPY FL C₁₆.
  • c.
    Resuspend the washed organoids in this solution (200 μL per well).
  • d.
    Transfer into a 48-well plate.

CRITICAL: Perform all steps from now on in the dark or under minimal light.

• 35.Incubate at 37°C for 20 min.
• 36.
  Stop the incubation and wash:

  • a.
    Add 400 μL ice-cold 0.2% BSA/PBS to each well to stop the incubation.
  • b.
    Quickly transfer the suspension into a 1.5 mL EP tube.
  • c.
    Centrifuge at 200 × g for 2 min at 4°C. Discard the supernatant.
  • d.
    Add 1 mL ice-cold PBS, gently pipette to resuspend, then centrifuge at 200 × g for 2 min at 4°C.
  • e.
    Discard the supernatant.
  • f.
    Repeat the wash (steps d–e) twice more.
• 37.
  Digest organoids into single cells:

  • a.
    Add 100 μL TrypLE to each tube, gently pipette 2–3 times.
  • b.
    Incubate at 37°C for 5–8 min.
• 38.
  Terminate digestion and wash:

  • a.
    Add 200 μL 10% FBS/PBS to stop digestion.
  • b.
    Centrifuge at 200 × g for 2 min at 4°C. Discard the supernatant.
  • c.
    Add 1 mL ice-cold PBS, gently pipette to resuspend, then centrifuge at 200 × g for 2 min at 4°C.
  • d.
    Discard the supernatant.
  • e.
    Repeat the wash (steps c–d) twice more.
• 39.Resuspend the single-cell pellet in 2% FBS/PBS.
• 40.Stain for viability:

 Before flow cytometry, add PI dye at a 1:1 ratio to label dead cells.

Note: After BODIPY FL C₁₆ incubation, keep all samples on ice and protected from light, and perform flow cytometry as soon as possible

• 41.Perform flow cytometry analysis on the prepared single-cell suspension to measure BODIPY FL C₁₆ fluorescence (505/512 nm) and PI staining (535/617 nm).

Note: Before loading, filter the single-cell suspension through a 70 μm cell strainer to remove undigested clumps and prevent clogging of the flow cytometer.

Protocol for assessing lipid accumulation in intestinal organoids

Timing: At least 7 h

Unlike fatty acid uptake assays, which measure the initial entry of exogenous fatty acids into cells, lipid accumulation assays quantify the net intracellular lipid content resulting from uptake, metabolism, and storage. Here, we develop and present a flow cytometry-based protocol to quantify lipid accumulation in dissociated small intestinal organoids, enabling evaluation of these processes in a physiologically relevant model. By preserving the cellular heterogeneity and structural organization of the intestinal epithelium, this approach enables quantitative assessment of intestinal lipid storage under conditions that more closely recapitulate the in vivo environment.

• 42.Use well-differentiated organoids cultured for 7–8 days for the assay.
• 43.
  Starve organoids in serum-free medium:

  • a.
    Remove complete medium.
  • b.
    Wash each well 2–3 times with PBS.
  • c.
    Add serum-free DMEM/F12 medium (300 μL per well).
  • d.
    Incubate for 4 h to complete starvation.
• 44.
  Remove Matrigel:

  • a.
    Aspirate the medium from the well.
  • b.
    Add 300 μL ice-cold PBS, leave for 30 s – 1 min.
  • c.
    Gently pipette the gel 1–2 times until it dissolves.
  • d.
    Transfer the suspension to a 1.5 mL EP tube.
  • e.
    Centrifuge at 100–200 × g for 2 min at 4°C. Discard the supernatant.
  • f.
    Add 1 mL ice-cold PBS, gently pipette to resuspend, then centrifuge again at 100–200 × g for 2 min at 4°C.
  • g.
    Repeat the wash (step f) twice more.

CRITICAL: Before incubating with BODIPY FL C₁₆, ensure Matrigel is completely removed.

• 45.
  Incubate with BODIPY FL C₁₆:

  • a.
    Dilute BODIPY FL C₁₆ stock solution to 0.5 μM in 0.1% BSA/PBS.
  • b.
    For blank controls, use 0.1% BSA/PBS without BODIPY FL C₁₆.
  • c.
    Resuspend the washed organoids in this solution (200 μL per well).
  • d.
    Transfer into a 48-well plate.

CRITICAL: Perform all steps from now on in the dark or under minimal light.

• 46.Incubate at 37°C for 30 min.
• 47.
  Stop the incubation and wash:

  • a.
    Add 400 μL ice-cold 0.2% BSA/PBS to each well to stop the incubation.
  • b.
    Quickly transfer the suspension into a 1.5 mL EP tube.
  • c.
    Centrifuge at 200 × g for 2 min at 4°C. Discard the supernatant.
  • d.
    Add 1 mL ice-cold PBS, gently pipette to resuspend, then centrifuge at 200 × g for 2 min at 4°C.
  • e.
    Discard the supernatant.
  • f.
    Repeat the wash (steps d–e) twice more.
• 48.
  Culture organoids without Matrigel:

  • a.
    Resuspend the organoids in complete medium (300 μL per well).
  • b.
    Transfer to a 48-well plate and culture at 37°C for no more than 6 h.
  • c.
    If reagent stimulation is required, add the reagent to the medium at this step.

Note: Do not culture Matrigel-free organoids for more than 6 h, as extended culture reduces viability. Avoid re-embedding organoids in Matrigel, because BODIPY FL C₁₆ excreted by cells may be retained in Matrigel and reabsorbed, causing inconsistent results.

• 49.
  Digest organoids into single cells:

  • a.
    Add 100 μL TrypLE, gently pipette 2–3 times.
  • b.
    Incubate at 37°C for 5–8 min.
• 50.
  Terminate digestion and wash:

  • a.
    Add 200 μL 10% FBS/PBS to stop digestion.
  • b.
    Centrifuge at 200 × g for 2 min at 4°C. Discard the supernatant.
  • c.
    Add 1 mL ice-cold PBS, gently pipette to resuspend, then centrifuge at 200 × g for 2 min at 4°C.
  • d.
    Discard the supernatant.
  • e.
    Repeat the wash (steps c–d) twice more.
• 51.Resuspend the single-cell pellet in 2% FBS/PBS.
• 52.Stain for viability:

 Immediately before flow cytometry, add PI dye (final concentration 1μg/mL) to label dead cells.

CRITICAL: After BODIPY FL C₁₆ incubation, keep organoids protected from light at all times.

• 53.Perform flow cytometry analysis on the prepared single-cell suspension to measure BODIPY FL C₁₆ fluorescence (505/512 nm) and PI staining (535/617 nm).

Note: Before loading, filter the single-cell suspension through a 70 μm cell strainer to remove undigested clumps and prevent clogging of the flow cytometer.

CRITICAL: Prepare an additional 1–2 wells following the fatty acid uptake protocol described above, with a 30 min BODIPY FL C₁₆ incubation instead of 20 min, omitting cytokine stimulation and the subsequent incubation period, to serve as a fluorescence baseline control.

Expected outcomes

The protocols described above provide an integrated workflow for measuring fatty acid uptake and intracellular lipid accumulation in intestinal epithelial cell lines and organoids using flow cytometry. In addition, we present a straightforward protocol for isolating and culturing mouse small intestinal organoids, enabling the study of lipid metabolism in a physiologically relevant model. Together, these protocols allow quantitative assessment of BODIPY FL C₁₆ uptake immediately after labeling and evaluation of intracellular lipid retention following a chase period, thereby distinguishing fatty acid absorption from lipid accumulation. Although optimized for intestinal epithelial cells and organoids, this workflow can be adapted to other cell types capable of lipid uptake with capacity after appropriate optimization.

In intestinal epithelial cell lines, incubation with BODIPY FL C₁₆ produces a readily detectable fluorescence signal compared with blank control cells incubated with 0.1% BSA/PBS alone (Figure 1B). Fluorescence intensity increases with both BODIPY FL C₁₆ concentration and incubation time (Figures 1B–1E). To maximize dynamic range while maintaining sensitivity to experimental perturbations, we used a 20 min pulse with 0.1 μM BODIPY FL C₁₆ in subsequent experiments; however, these parameters should be optimized for each cell type. Under these conditions, IL-22 treatment reduces BODIPY FL C₁₆ fluorescence, consistent with decreased fatty acid uptake⁴ (Figures 1F–1H). Following a 2 h or 24 h chase after the BODIPY FL C₁₆ pulse, fluorescence intensity progressively decreases as intracellular lipids are metabolized or exported, whereas the remaining fluorescence represents retained intracellular lipid (Figures 2B–2D). In contrast, treatment with lomitapide, an inhibitor of microsomal triglyceride transfer protein that blocks chylomicron assembly and lipid secretion, increases intracellular fluorescence during the chase period, consistent with enhanced lipid retention (Figures 2E–2G).

Figure 1.

Lipid uptake assay in Caco-2 cells

(A) Gating strategy for flow cytometry.

(B and C) Optimization of BODIPY FL C₁₆ concentration.

(D and E) Optimization of BODIPY FL C₁₆ incubation time.

(F–H) Effect of IL-22 on fatty acid uptake. The significance of the differences (C, E, G, and H) was determined using one-way ANOVA. Data are represented as mean ± SEM; ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 2.

Lipid accumulation assay in Caco-2 cells

(A) Gating strategy for flow cytometry.

(B–D) Time-course of lipid accumulation.

(E–G) Effect of lomitapide on lipid accumulation. Cells were treated with 25 μg/mL lomitapide. The significance of the difference (C, D, F, and G) was determined using one-way ANOVA. Data are represented as mean ± SEM; ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

During intestinal organoid culture, isolated crypts initially form compact spheroids that, over 1–7 days, develop budding structures, yielding mature intestinal organoids suitable for functional analysis (Figure 3). Following incubation with BODIPY FL C₁₆, organoids display robust fluorescence compared with blank controls (Figure 4B). IL-22 treatment decreases fluorescence intensity, indicating reduced fatty acid uptake (Figures 4C and 4D). After a 6 h chase period, fluorescence intensity declines substantially, while the remaining signal reflects intracellular lipid retention (Figures 5B–5D).

Figure 3.

Time-course morphology of intestinal organoids from day 0 to day 7

(A) Time-course images of intestinal organoid growth (days 0, 1, 3, 5, and 7); scale bars in all panels of Figure 3 represent 500 μm.

Figure 4.

Lipid uptake assay in intestinal organoids

(A) Gating strategy for flow cytometry.

(B) Fluorescence intensity of cellular BODIPY FL C₁₆ uptake.

(C and D) Effect of IL-22 on organoid lipid uptake. The significance of the difference (D) was determined using one-way ANOVA. Data are represented as mean ± SEM; ∗p < 0.05 and ∗∗∗p < 0.001.

Figure 5.

Lipid accumulation assay in intestinal organoids

(A) Gating strategy for flow cytometry.

(B–D) Time-course of lipid accumulation.

Limitations

This protocol uses flow cytometry to quantify fatty acid uptake and intracellular lipid accumulation in intestinal epithelial cells and organoids with high throughput and single-cell resolution. However, unlike imaging-based approaches such as confocal microscopy, flow cytometry does not provide spatial information on intracellular lipid distribution. Consequently, it cannot determine the subcellular localization of the fluorescent signal or distinguish whether labeled lipids reside in the cytoplasm, endoplasmic reticulum, mitochondria, or lipid droplets.

BODIPY FL C₁₆ is a fluorescent fatty acid analog and may not fully recapitulate the uptake, intracellular trafficking, and metabolic fate of endogenous long-chain fatty acids. In addition, intracellular fluorescence can be influenced by probe efflux, metabolic conversion, or intracellular processing, which should be considered when interpreting fluorescence intensity as a measure of lipid uptake or accumulation.

Sample preparation may also introduce variability. Enzymatic dissociation, repeated washing, and centrifugation can reduce fluorescence intensity, induce cellular stress, or preferentially affect fragile cell populations. Because BODIPY FL C₁₆ is light sensitive, all samples should be protected from light and analyzed promptly after staining to minimize fluorescence loss and ensure reproducible results.

For intestinal organoid lipid accumulation assays, organoids are cultured in suspension without Matrigel during the incubation and chase periods to facilitate reagent access and flow cytometric analysis. Under these conditions, organoid viability is limited, thereby restricting the duration of cytokine stimulation or chase experiments and reducing the protocol’s suitability for investigating slower lipid metabolic processes.

Overall, this protocol is well suited for quantitative, population-level analysis of fatty acid uptake and intracellular lipid accumulation but is not designed to assess subcellular lipid localization, intracellular trafficking dynamics, or lipid metabolism within intact tissue architecture.

Troubleshooting

Problem 1

For steps 12 and 41 of the cell uptake assay, the control group’s fluorescence intensity varied considerably across biological replicates.

Potential solution

The variability likely arises from either inadequate pre-starvation washing, which leads to inconsistent uptake of BODIPY FL C₁₆ and can be addressed with 2–3 ice-cold PBS rinses, or insufficient mixing of the BODIPY/BSA solution, which creates local concentration gradients and can be resolved by thorough vortexing or shaking prior to use.

Problem 2

In steps 33 and 44, a considerable number of organoids are lost during dissociation/release from the Matrigel matrix, leading to poor recovery.

Potential solution

To minimize organoid loss, the organoid-Matrigel mixture should be kept on ice throughout the procedure to reduce cell damage during matrix degradation, and low-adsorption pipette tips should be used to prevent organoid adhesion to the tip walls. In addition, to preserve organoid morphology and integrity, forceful or repetitive pipetting must be avoided; instead, gentle and slow pipetting with wide-bore tips is strongly recommended to minimize mechanical shearing forces. In addition, all tips were pre-coated with 0.1% BSA in PBS before use. More specifically, the tip was used to aspirate and expel the BSA solution three times. The BSA solution was then discarded, while the tip (now coated with a protein layer on its inner surface) was immediately used for organoid transfer. This coating effectively blocks non-specific binding sites and minimizes sample loss.

Problem 3

In step 53, considerable variability in fluorescence intensity is observed across biological replicates of the control group, compromising data reliability and reproducibility.

Potential solution

To address this issue, it is first recommended to ensure uniform initial fluorescence intensity across all samples by collecting all organoids, incubating them together with the BODIPY/BSA solution, then re-collecting and thoroughly mixing before distributing the organoid suspension into culture plates for subsequent procedures. If the inter-replicate variability persists after this optimization, it may be attributed to incomplete removal of the Matrigel, as residual matrix components could interfere with uniform probe uptake by organoids. In such cases, a refined dissociation procedure is suggested: insert the front end of a 1000-μL pipette tip into the rear opening of a 10-μL tip, allowing the fine tip of the smaller one to extend forward as an elongation of the larger one, thereby creating a narrow-tipped assembly that enables precise aspiration of residual Matrigel and ensures thorough removal, ultimately reducing variability among replicates.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Li-Hao Huang (lihao_huang@fudan.edu.cn).

Technical contact

Technical questions about executing this protocol should be directed to and answered by the technical contacts, Jiaqi Wu (1007163108@qq.com) and Yu Gu (21110700015@m.fudan.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new datasets or code.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (32371243, 32571372, 32350610248, and 32171168 to L.-H.H.).

Author contributions

L.-H.H. supervised the research, designed the experiments, and finalized the manuscript. J.W. performed Caco-2 and organoid lipid accumulation experiments. Y.G. provided significant support for the separation of intestinal crypts and the cultivation of small intestinal organoids. S.W. performed Caco-2 and organoid lipid uptake experiments. J.W. and S.L. drafted the manuscript together. All authors have read and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.