A Protocol for Size-Based Enrichment of Lipid Droplets from Bovine Mammary Epithelial Cells and Mammary Tissues

Summary

This protocol aims to achieve size-based separation and enrichment of lipid droplets via sequential centrifugation, enabling accurate, reproducible investigation of lipid metabolism mechanisms and lipid droplet-associated biological processes.

Abstract

This protocol describes a sequential differential centrifugation approach to enrich lipid droplets (LDs) of different sizes from bovine mammary epithelial cells (BMECs) and mammary tissues, enabling size-resolved analysis beyond conventional bulk LD isolation methods. As dynamic regulatory hubs of intracellular lipid metabolism, LDs serve critical functions by storing neutral lipids (such as triglycerides and cholesteryl esters) and coordinating their synthesis, hydrolysis, and transport. They play a central role in maintaining energy homeostasis, supporting membrane biogenesis, and facilitating cellular signal transduction. LD extraction is essential for investigating the regulatory mechanisms of lipid metabolism. Studying mammary gland lipid metabolism has significant implications for infant development, human health, agricultural economics, and fundamental cell biology. Therefore, we describe a differential centrifugation method for extracting LDs from BMECs and mammary gland tissue. In contrast to conventional bulk LD isolation approaches, this protocol enables size-based enrichment of LD subpopulations by sequentially adjusting centrifugal forces. The integrity and relative enrichment of LD fractions are preliminarily evaluated using BODIPY493/503 staining, providing a practical and reproducible approach for downstream analyses of lipid metabolism and LD-associated processes.

Introduction

Lipid droplets (LDs) ​are dynamic organelles within cells that serve as primary storage sites for neutral lipids¹. Abnormal accumulation of LDs is associated with a wide range of diseases, extending beyond obesity to include conditions such as fatty liver disease², cardiovascular diseases³, diabetes⁴, and cancer⁵. Conversely, an insufficient number of LDs can also have severe consequences, leading to the loss of cellular energy reserves and subsequent functional impairments⁶. In the context of agricultural economics, insufficient LDs in ruminants can result in inadequate milk fat secretion, giving rise to milk fat depression, which in turn compromises the nutritional value and marketability of dairy products⁷. Currently, the regulation of LD metabolism has emerged as a significant area of research.

The development of extraction techniques for LDs since the 20th century has consistently aimed to extract LDs in greater quantities, with higher purity and better preserved biological activity. This involves the continuous establishment and optimization of methodologies. Currently, several companies have designed commercial kits for LD separation⁸. Although these kits greatly reduce the separation time and only requires the use of conventional centrifuges, for samples that are difficult to dissociate, the reagent cannot be adjusted, and it is difficult to optimize the test steps. Furthermore, the extraction of LDs plays an indispensable role in elucidating disease mechanisms, including those of metabolic diseases such as fatty liver⁹, cancer¹⁰, and neurodegenerative diseases¹¹, serving as a multidimensional probe for deciphering pathological pathways. The protein and lipid compositions can also be dissected by extracting LDs to reveal the diversity of LDs biological functions¹².

The formation of milk fat is not merely a process of substrate degradation; rather, it represents a highly orchestrated lipid synthesis and packaging process carried out by mammary epithelial cells.¹³. The generation of milk fat in dairy cows relies on the apical membrane secretion mechanism of these cells: triglycerides synthesized in the endoplasmic reticulum form cytoplasmic LDs, which are then transported to the apical membrane and secreted via budding. During this process, LDs are enveloped by a milk fat globule membrane derived from the apical membrane, resulting in the formation of structurally stable milk fat globules¹⁴. The bovine mammary gland serves as a classic model for studying lactation in mammals, and the core mechanisms governing LD synthesis and secretion are highly conserved in humans¹⁵. Establishing methods to extract LDs from both BMECs and mammary tissue can bridge livestock production and human health. Proteomic and lipidomic analyses of the extracted LDs can subsequently be leveraged to improve milk yield and quality, ensuring safety and security.

The present method is primarily based on the low-density characteristic of LDs, which causes them to float to the top of the gradient during centrifugation¹⁶. The extraction procedure consists of three main steps: sample collection, ultracentrifugation for LD flotation, and LD washing. Sucrose (2.5 M) is added to the buffer to protect the LDs, the centrifugation speed is then adjusted according to experimental requirements; typically, large LDs can be enriched at forces below 10,000 x g, while higher speeds (above 10,000 x g) are employed to extract small LDs, as larger LDs may rupture under these conditions. Finally, the extracted LDs undergo multiple washes to prevent contamination, and the extraction purity is preliminarily assessed using BODIPY493/503 staining.

Conventional LD isolation methods primarily achieve bulk recovery of heterogeneous LD populations, limiting analysis of size-dependent structural and functional differences. The present protocol extends standard approaches by enabling size-based enrichment of LD subpopulations through sequential differential centrifugation, facilitating investigation of LD growth, turnover, intracellular trafficking, and differential utilization by lipolytic or autophagic pathways. This approach achieves enrichment rather than absolute purification, and partial overlap between size fractions may occur due to continuous size distributions and shared physical properties.

Protocol

Determine target lipid droplet size for enrichment (Figure 1).

1. Reagent setup

1. PBS: Weigh 8 g NaCl, 0.2 g KCl, 3.58 g Na₂HPO₄·12H₂O, and 0.24 g KH₂PO₄ into 800 mL ddH₂O. Adjust pH to 7.4 with HCl, mix thoroughly, and bring volume to 1 L. Store at 4 °C for up to 1 year after autoclaving.
2. Sucrose (2.5 M): Weigh 856 g of sucrose and dissolve it in 400 mL of ddH₂O. Stir thoroughly until the sucrose is completely dissolved, then adjust the volume to 1 L. Autoclave the solution and store at 4 °C for up to 3 months.
3. Buffer A: Weigh 1.79 g of Tricine and dissolve it in 400 mL of ddH₂O. Adjust pH to 7.8 with KOH. After pH adjustment, add 50 mL of pre-prepared 2.5 M sucrose solution. Mix thoroughly and adjust the volume to 500 mL. After autoclaving, the buffer can be stored at 4 °C for up to 3 months.
4. Buffer B: Weigh 0.95 g HEPES, 1.49 g KCl, and 0.038 g MgCl₂·6H₂O separately in 180 mL ddH₂O. Adjust pH to 7.4 with KOH, mix thoroughly, and bring volume to 200 mL. After autoclaving, store at 4 °C for up to 3 months.
5. PMSF (1,000x, 0.2 M): Weigh 0.175 g of PMSF and dissolve it in 5 mL of DMSO. Store protected from light at -20 °C for up to one year.
   NOTE: PMSF is highly toxic; appropriate protective measures must be taken. During preparation, ensure complete protection from light; PMSF is difficult to dissolve and can be aliquoted into 1.5 mL microcentrifuge tubes.

2. Extraction of LDs from cells

1. BMECs culture
   1. Retrieve five tubes of frozen BMEC cells from the -80 °C refrigerator and thaw them rapidly in a 37 °C water bath. Centrifuge the tubes at 250 x g for 4 min 30 s after complete thawing.
   2. Discard the cryopreservation medium with a pipettor, add 1 mL of 10% FBS to resuspend the cells, mix and gently add the cells to a 100-mm cell culture dish containing 6 mL of 10% FBS. Gently rock the dish to ensure even cell distribution and incubate at 37 °C with 5% CO₂.
   3. Monitor cell morphology under a microscope and perform subculturing when cells reach 80%–90% confluency.
      NOTE: Ensure optimal cell condition and sufficient cell number prior to LD extraction to improve yield and reproducibility.
   4. Discard the original culture medium in the petri dish with a pipette, and add 1 mL PBS along the wall of each dish. After washing twice, add 1 mL trypsin to each dish and incubate at 37 °C for 3 min 30 s.
   5. Observe whether the cells are completely digested under the microscope, add 1 mL of 10% FBS to terminate the digestion after the digestion is complete. Gently resuspend the cells by pipetting, transfer them to a 2 mL microcentrifuge tube, and centrifuge at 250 x g for 4 min 30 s.
   6. Discard the supernatant and repeat the procedures described in Step 1.2.
   7. Expand cells using approximately 30 culture dishes or larger culture vessels, or use larger culture dishes for cell culture. Add 150 µmol/L t10,c12-CLA (diluted in DMEM) to treat cells the night before extraction. Use 7 mL of culture medium per 100 mm culture dish. The culture time depends on the size of LDs that need to be separated.
      NOTE: Based on prior optimization, treat BMECs with t10,c12-CLA for 6 h to enrich large LDs or for 12–24 h to enrich small LDs. Since cells contain few LDs, exogenous fatty acids are usually added to induce the accumulation of LDs. In this study, add t10,c12-CLA to cultured cells to induce the accumulation of LDs.
2. Sample collection
   1. Discard the original medium, add 1 mL of ice-cold PBS and wash 3 times. Do not discard PBS during the final wash. Using 30 dishes of 100-mm cells as an example, collect cells from every 10 plates into one 15 mL centrifuge tube, each tube contains approximately 1x108, resulting in a total of 3 centrifuge tubes.
      NOTE: Maintain all samples at ≤4 °C throughout the extraction process to prevent protein degradation in LDs.
   2. After balancing, centrifuge at 1,000 x g, 4 °C for 10 min.
   3. Discard the PBS. Add 10 mL of pre-prepared Buffer A and 10 µL of PMSF (1,000x, 0.2 M) to the centrifuge tube. Mix thoroughly and incubate on ice for 20 min.
3. Cell lysis and initial fractionation
   1. Disrupt cells using a probe-type ultrasonic homogenizer operated in pulsed cycles of 5 s ON and 10 s OFF, resulting in a total effective sonication time of 10 min over a 30 min period. Set the instrument to 40% power (corresponding to a probe amplitude of approximately 76 µm) using a 3 mm diameter probe.
      NOTE: Keep samples on ice throughout the procedure to prevent overheating.
   2. Centrifuge the homogenate at 3,000 x g and 4 °C for 10 min to remove nuclei and cellular debris. Collect the supernatant as the post-nuclear supernatant (PNS).
4. Ultracentrifugation of floating LDs
   1. Filter the supernatant through a 100 µm cell strainer.
   2. Pipette 10 mL of PNS into a 15 mL centrifuge tube. Add 2 mL of sucrose (2.5 M) and 2 mL of Buffer B sequentially to the top of the PNS (the sucrose volume may be reduced appropriately while increasing the Buffer B volume to enhance LD purity), and then centrifuge for the first time (5,000 x g, 4 °C, 20 min).
   3. After centrifugation, collect the milky, cream-like LD layer at the top of the tube. To collect LDs, first add 100 µL of Buffer B to a 1.5 mL microcentrifuge tube, then use a 20 µL pipette to gently aspirate the floating LD layer multiple times, collecting as many LDs as possible.
      NOTE: LDs form a floating cream layer at the top; avoid aspirating the underlying solution as much as possible to prevent contamination.
   4. Perform a second centrifugation (10,000 x g, 4 °C, 30 min) and collect the LDs.
   5. Transfer the solution to an ultracentrifuge tube compatible with a swinging-bucket rotor, cap it, and balance the tubes. Perform the third centrifugation in an ultracentrifuge (50,000 x g, 4 °C, 40 min) and collect the LDs.
      NOTE: Precisely balance tubes (to 0.0001 g) to ensure safe ultracentrifuge operation.
   6. After balancing, perform the fourth centrifugation (150,000 x g, 4 °C, 50 min) to continue collecting LDs. Optimize speed and duration according to target LD size.
      NOTE: Prolonged or repeated high-speed centrifugation may disrupt LDs. In contrast, short-duration centrifugation at 10,000 x g during washing primarily removes impurities and has minimal impact on LD integrity. This method is adaptable; optimization may be required for different cell types or tissues.
5. Washing LDs
   1. Resuspend the collected LDs in cold Buffer B and centrifuge at 10,000 x g and 4 °C for 5 min.
   2. Carefully remove the supernatant and repeat the washing step at least four times using the same buffer volume, centrifugal force, and centrifugation time. For small LDs, increase the centrifugal force as needed to ensure efficient separation.
   3. After the final wash, pool the LD fractions, mix gently, and store at −80 °C.
      ​NOTE: Do not omit washing steps, even though some LD loss is inevitable. Continue washing until no visible membrane debris remains.

3. Extraction of LDs from tissue

1. Sample collection
   1. Retrieve bovine mammary tissue samples from liquid nitrogen. Weigh out 5 g of tissue and thaw it on ice until no ice crystals remain.
   2. Rinse the tissue with PBS or physiological saline.
   3. Mince the tissue into 1-2 mm pieces using surgical scissors.
   4. Transfer the minced tissue into a pre-chilled mortar containing 12 mL of Buffer A and 10 µL of PMSF (1,000x, 0.2 M). Grind the mixture on ice for approximately 10 min.
   5. Incubate the homogenate on ice for 20 min.
   6. After thorough grinding, collect the homogenate into a 15 mL centrifuge tube.
   7. Centrifuge at 2,500 x g, 4 °C for 10 min. The resulting supernatant is PNS.
2. Ultracentrifugation of floating LDs
   1. Filter the supernatant through a 100 µm cell strainer.
   2. Pipette 10 mL of PNS into a 15 mL centrifuge tube. Add 2 mL sucrose and 2 mL Buffer B sequentially. Perform the first centrifugation (4,000 x g, 4 °C, 20 min). Collect the milky, cream-like material.
   3. Perform a second centrifugation (7,000 x g, 4 °C, 30 min) and collect the LDs.
   4. Perform a third centrifugation (10,000 x g, 4 °C, 40 min) and collect the LDs.
   5. Transfer the supernatant to an SW40 tube and perform ultracentrifugation (50,000 x g, 4 °C, 50 min). Collect the LDs.
   6. Perform a fifth centrifugation (100,000 x g, 4 °C, 60 min). Collect the LDs.
3. Washing LDs
   Refer to the washing method for cell-extracted LDs.

4. Preliminary validation of LD purity by staining

1. Prepare 10 µg/mL neutral lipid fluorescent dye solution in advance under light-protected conditions (BODIPY: Buffer B = 1:100, V/V).
2. Add an appropriate amount of mixed LDs and Buffer B to a microcentrifuge tube. Under light-protected conditions, add an appropriate amount of neutral lipid fluorescent dye solution. Mix thoroughly and incubate for 10 min.
3. Prepare a clean microscope slide. Dispense 2.5 µL of the mixture onto the coverslip. Gently place the slide over the mixture, avoiding air bubbles. Observe under an inverted fluorescence microscope.

5. Validation of LD purity by Western blot

1. Protein concentration of LD fractions was measured using a BCA protein assay kit.
2. For Western blot analysis, mix 20 µg of protein from each sample with 5x SDS loading buffer and heat at 99 °C for 10 min to ensure complete denaturation.
3. Then, separate the proteins by SDS–PAGE on 10% gradient polyacrylamide gels according to their molecular weights.
4. After electrophoresis, transfer the proteins onto pre-activated PVDF membranes, then block with 5% BSA at room temperature for 1.5 h. Subsequently,iIncubate the membranes overnight at 4 °C with primary antibodies, including rabbit anti-Perilipin 2 (1:500) and rabbit anti-β-actin (1:5000).
5. Wash the membranes three times with 1x TBST for 10 min each, then incubate with HRP-conjugated goat anti-rabbit secondary antibody (1:5000) for 1.5 h at room temperature.
6. After three additional washes with TBST, detect protein bands using an enhanced chemiluminescence (ECL) kit and visualize with a gel imaging system.

6. Pause points and storage guidelines

1. Keep PNS on ice for up to 1–2 h prior to ultracentrifugation.
2. Keep collected LDs on ice for up to 1–2 h before washing. For short-term storage, store LDs at 4 °C for up to 24 h. For long-term storage, store LDs at −80 °C and limit freeze–thaw cycles to no more than one to preserve LD integrity.
3. During all experimental pauses, keep samples on ice with tube caps closed to minimize contamination.

7. Troubleshooting

1. Thin floating layer: adjust buffer volume or centrifugation force.
2. Poor floatation: increase centrifugation speed or extend time.
3. Contamination: ensure sterile handling and filtered buffers.
4. LD rupture: keep samples on ice, reduce centrifugation force, or resuspend gently.

Results

Workflow and methodological framework
A decision-tree schematic (Figure 1) was established to guide the selection of centrifugation conditions according to experimental objectives. Specifically, centrifugal forces ≤10,000 x g were used to enrich large LDs (≥2 µm), whereas forces >10,000 x g were applied to enrich small LDs (<1 µm). This framework provides a practical basis for achieving size-based LD separation. The overall workflow for LD isolati...

Discussion

LDs are unique organelles characterized by a neutral lipid core enclosed by a phospholipid monolayer. They bud from the endoplasmic reticulum and dynamically change their size and number, facilitating intracellular transport and extensive communication with other subcellular structures to coordinate cellular lipid homeostasis^17,18,19. This dynamic behavior is further modulated by systemic metabolic cues, as insulin has been show...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco	25200072	reagent
10% PAGE Gel Rapid Preparation Kits	Beijing BIOMAN	PG112	reagent
100 μm cell strainer	Beyotime	FSTR100	reagent
100-mm cell culture dish	Corning	490167	reagent
Amersham Imager 600	GE Healthcare Life Sciences	600	equipment
Autoclave	Panasonic	MLS-3751L	equipment
BCA protein assay kit	Beijing Dingguo.	BCA01	reagent
BODIPY 493/503	Invitrogen	D3922	reagent
Centrifuge	Eppendorf		equipment
DMEM	HyClone	SH30022.01	reagent
DMSO	Solarbio	D8370	reagent
Fetal bovine serum	Gibco	2492319	reagent
HEPES	MedChemExpress	HY-D0857	reagent
HRP-conjugated AffiniPure goat anti-rabbit secondary antibody	Proteintech	SA00001-2	reagent
KCl	HUSHI	20030666	reagent
KH2PO4	HUSHI	10017608	reagent
MgCl2	HUSHI	20059028	reagent
Na2HPO4·12H2O	HUSHI	S112623	reagent
NaCl	HUSHI	10019328	reagent
Omni-Easy Instant Protein Loading Buffer	Shanghai Epizyme Biomedical Technology	LT101S	reagent
Omni-ECL Ultra-Sensitive Chemiluminescence Detection Kit	Shanghai Epizyme Biomedical Technology	SQ201	reagent
PBS	HyClone	SH30256.01	reagent
Penicillin & Streptomycin 100×	NCM Biotech	CLOOC5	reagent
pH meter	METTLER TOLEDO	S220K	equipment
Pipette	Eppendorf		equipment
PMSF	Coolaber	CP8651	reagent
PVDF Membrane Transfer Kit	GenScript	eBlot L1	reagent
Rabbit anti-Perilipin 2	Abcam	15294-1-AP	reagent
Rabbit anti-β-actin	Proteintech	20536-1-AP	reagent
Research grade inverted fluorescence microscope	Olympus	IX73	equipment
Sucrose	Solarbio	S8271	reagent
SW 40 Ti Rotor	BECKMAN COULTER	331301	equipment
Trans-10, cis-12 conjugated linoleic acid	CAYMAN	90145	reagent
Tricine	Coolaber	CT11331	reagent
Ultracentrifuge	BECKMAN COULTER	Optima XE-100	equipment
Ultrasonic cell crusher	SCIENTZ	JY92-11N	equipment