Materials and Reagents

µ-dish cell culture imaging dish, 35 mm, high (Ibidi, catalog number: 81156)
1,000, 200, and 20 µL pipette tips (pre-sterile w/ filter, hinged racks) (ExpellPlus, catalog numbers: 5030150C, 5030090C, 5130062C)
15 mL centrifuge tubes (Corning, catalog numbers: 430791)
Corning^® Costar^® Stripette^® serological pipettes, individually paper/plastic wrapped, 5 and 10 mL (Corning, catalog numbers: CLS4487, 4488)
Falcon^® 100 mm TC-treated cell culture dish (Corning, catalog number: 353003)
RPMI 1640 media (Lonza, catalog number: BE 12-115F)
Fetal bovine serum (FBS) (Gibco^TM, catalog number: 10270106)
Penicillin-streptomycin mixture (Lonza, catalog number: DE17-603E)
Dulbecco's phosphate buffered saline (PBS) (10×), 95 mM (PO₄) without calcium or magnesium (Lonza, catalog number: BE17-515Q)
Bovine serum albumin (BSA)-oleate monounsaturated fatty acid complex (5 mM) (Cayman Chemical, catalog number: 29557)
BSA control for BSA-fatty acid complexes (5 mM) (Cayman Chemical, catalog number: 29556)
Trypsin/EDTA solution (Lonza, catalog number: CC-5012)
Complete RPMI growth medium (see Recipes)
Starving medium (see Recipes)

Biological materials

Cell line: MH-S (ATCC number: CRL-2019^TM)

Equipment

Rainin Pipet-Lite XLS (Mettler Toledo, models: SL1000 and SL200)
Forma Direct heat CO₂ incubator 184 L digital model (Thermo Scientific, model: 311; TC 230)
Biosafety cabinet (Telstar, model: Bio II Advance Plus IV)
Centrifuge Sorvall ST 16R (Thermo Scientific, model: 75004380)
3D Cell Explorer microscope (Nanolive, Ecublens, Switzerland)
CellDrop^TM FL automated cell counter (DeNovix)
Laboratory water bath (Memmert, model: WNB 14)

Software

Steve software v1.6.3496 (Nanolive, Ecublens, Switzerland)
ImageJ (FIJI) software (https://imagej.net/software/fiji/downloads)

Procedure

Cell culture
1. Grow MH-S cells inside of a humidified incubator with 5% CO₂ at 37 °C in complete RPMI growth medium (see Recipes) to 90% confluency in a 100 mm dish.
2. Remove the culture media and wash the cells with 10 mL of PBS 1×.
3. After removing the PBS, add 2 mL of trypsin/EDTA solution to detach cells, incubate in a humidified incubator with 5% CO₂ for 5 min at 37 °C, and add 8 mL of complete RPMI growth medium to stop the reaction.
  Note: Temper the growth medium and trypsin solution at 37 °C in a water bath.
4. Collect the cells and centrifuge at 423 × g for 5 min. Remove supernatant and resuspend the pellet in 5 mL of complete RPMI growth medium.
5. Calculate the cell concentration using an automated cell counter.
6. Seed MH-S cells in 35 mm μ-dishes (3 × 10⁵ cells per dish) in 1.5 mL of complete RPMI growth medium.
  Note: Remember to seed additional dishes for control condition (see step B3).
7. Incubate cells at 37 °C in a humidified incubator with 5% CO₂ overnight.
8. Temper the starving medium (see Recipes) at 37 °C in a water bath.
9. Change the cells to tempered starving medium and incubate under the same culture conditions for approximately 3 h.

BSA-oleate treatment
1. Defrost an aliquot of BSA-oleate monounsaturated fatty acid complex (5 mM) in the water bath for 2 min.
2. Prepare the BSA-oleate working solution at 100 µM in starving medium.
  Note: For 1.5 mL of medium, you need 60 µL of BSA-oleate monounsaturated fatty acid complex.
3. Change the starving medium to starving medium + BSA-oleate (100 µM) or starving medium + BSA for the control.
4. Prewarm the stage of the 3D Cell Explorer microscope to 37 °C at least 30 min before starting the acquisition.
5. Place the plate in the 3D Cell Explorer microscope stage where the incubation conditions (37 °C and 5% CO₂) will be maintained during the rest of the experiment.
  Note: Other holotomographic microscopes can also be used.

Image acquisition
This protocol is customized for a 3D Cell Explorer microscope equipped with a 60× magnification dry objective (λ = 520 nm, sample exposure 0.2 mW/mm²) and a depth of field of 30 μm.
1. Proceed to capture images every minute for 18 hours, using the Steve software that controls the microscope.
2. Each image provides a reconstruction of 30 μm in the z-axis, which corresponds to 100 images. Most of the images from the 100-image stacks are in fact out of focus, because samples are only visualized in a small range in the z-axis (around 5–10 stacks). From the whole z-stack, select at each time point the z-image in which the focus and resolution of the cells is best observed. LD are observed as intracellular spheres that have a high RI.
3. Export the image using Steve software to transform RI volumes into .tiff format. Pictures are exported by default as 32-bits images.
4. Process the exported image (.tiff format) using the FIJI software for performance purposes. Transform images to 8-bits and adjust gamma, brightness, and contrast identically for compared image sets using Fiji software.
5. An example of time-lapse refractive images of LD in alveolar macrophages (MH-S cells) is shown in Figure 1.
  
  Figure 1. Time-lapse refractive images of lipid droplets (LD) in alveolar macrophages (MH-S cells). Images show LD accumulation in MH-S cells treated with oleic acid (green) compared to control condition (blue). Close-up images shown at the right column correspond to the position indicated in the small squares.

Recipes

Complete RPMI growth medium
RPMI 1640 media supplemented with 10% FBS and 1% penicillin-streptomycin.

Starving medium
RPMI 1640 media supplemented with 2% FBS and 1% penicillin-streptomycin.

Acknowledgments

We are grateful to Instituto de Salud Carlos III for financial support to S.H.(PI20CIII/00018). A. Pérez-Montero is supported by Comunidad de Madrid (PEJ-2020-AI/BMD-17651).

Competing interests

The authors declare no competing interests.

References

Agudelo, C. W., Samaha, G. and Garcia-Arcos, I. (2020). Alveolar lipids in pulmonary disease. A review. Lipids in health and disease 19(1): 122-122.
van Dierendonck, X., Vrieling, F., Smeehuijzen, L., Deng, L., Boogaard, J. P., Croes, C. A., Temmerman, L., Wetzels, S., Biessen, E., Kersten, S., et al. (2022). Triglyceride breakdown from lipid droplets regulates the inflammatory response in macrophages. Proc Natl Acad Sci U S A 119(12): e2114739119.
Xu, S., Zhang, X. and Liu, P. (2018). Lipid droplet proteins and metabolic diseases. Biochim Biophys Acta Mol Basis Dis 1864(5, Part B): 1968-1983.
Sandoz, P. A., Tremblay, C., van der Goot, F. G. and Frechin, M. (2019). Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux. PLoS Biol 17(12): e3000553.