Materials and Reagents

Pipette tips (Thermo Fisher Scientific, different sizes and types)
Centrifuge tube, 5-ml round-bottomed (Sangon Biotech, catalog number: F610888)
Cryogenic microtubes, 1.5-ml deep cap, conical-bottomed, sterile (Sangon Biotech, catalog number: F600154)
Gel blotting paper (Sangon Biotech, catalog number: F513323)
Yeast extracts (OXCID, catalog number: LP0021)
Peptone (BD, catalog number: 211677)
D-glucose (Sangon Biotech, catalog number: A501991)
Yeast nitrogen base without amino acids and ammonium sulfate (Sangon Biotech, BBI, catalog number: A600505)
Chloroform (General-Reagent, catalog number: G75915B)
Methanol (General-Reagent, catalog number: G75851B)
Coomassie Brilliant Blue R-250 (Sangon Biotech, catalog number: CB0037)
Hexane (General-Reagent, catalog number: G14153D)
Diethyl ether (HUSHI, catalog number: 10009318)
Acetic acid (General-Reagent, catalog number: G73562B)
Lipid standard: 18:1 DG (1,2-dioleoyl-sn-glycerol) 2 mg/ml chloroform (Avanti, catalog number: 800811C)
Lipid standard: Egg PC (L-alpha-phosphatidylcholine) 10 mg/ml chloroform (Avanti, catalog number: 840051C)
TLC Silica gel 60 (25 aluminium sheets 20 × 20cm; Merk Millipore, catalog number: 105553)
Glass thin-layer chromatography (TLC) developing chambers (100 × 100, generic)
Glass beads (40 or 50 mesh, generic)

Equipment

Pipettes (Thermo Fisher Scientific, different types)
Shaking incubator (CRYSTAL, model: IS-RDH1)
Spectrophotometer (Shanghai Jing-Hua, model: 722S)
Centrifuge (Thermo, model: SORVALL ST16R)
Freeze-dryer/lyophilizer (Labconco, model: Freezone 6 Plus)
Analytical scale (Shanghai Heng-Ping, model: FA2104)
Drying oven (BOXUN, model: GZX-9146MBE)
Bead mill (Shanghai Jing-Xin, model: Tissue lyser-96)
Thermomixer (ALLSHENG, model: MSC-100)
TLC developing chamber with cover (generic)
Gel imaging system (Bio-Rad, model: Gel Doc 721BR05189)

Software

ImageJ (https://imagej.net/Downloads)

Procedure

Cell growth and harvest
1. Grow yeast cells in 100 ml YPD medium (1% yeast extract, 2% peptone, 2% glucose) overnight in a 30°C shaking incubator (250 rpm) until the optical density (OD₆₀₀) of the culture is about 0.7-1.0.
2. Harvest the cells by centrifugation, 10,000 × g for 5 min at room temperature. Remove the supernatant completely and discard.
3. Freeze-dry the cells in a freeze-dryer and store at -80°C.
Total lipid extraction
1. For each freeze-dried sample, transfer 0.25 mg into a 1.5-ml cryogenic microtube.
2. Add 200 μl washed glass beads to each sample tube.
3. Add 500 μl chloroform-methanol (2:1, v/v) to each sample tube. Screw on the Teflon-sealed cap tightly.
4. Place the sample tubes in a bead mill and perform mechanical shearing at maximum speed for 5 min at room temperature. Repeat the mechanical lysis twice; rearrange tube locations in-between to avoid uneven cell lysis (Step 1 in Figure 1).
5. Centrifuge the cell lysates at room temperature, 10,000 × g for 5 min. After centrifugation, cell debris will be sandwiched between the supernatant and the glass beads (Step 2 in Figure 1). Transfer the supernatant to new tubes for collection (Step 3 in Figure 1).
6. Repeat the chloroform-methanol extraction procedure (Steps 2-6 in Figure 1) five times.
7. Merge the supernatant collections of each sample in a 5-ml tube and evaporate on a compact thermomixer at 60°C overnight with the lid off. This step needs to be performed in a fume hood. After evaporation, membrane-like substances will be left at the bottom of the tubes.
  
  Figure 1. Schematic depiction of the lipid extraction procedure
Lipid analysis by TLC
1. Dissolve the lipid extracts in 100-500 μl chloroform.
2. Prepare the solvent mixtures:
  1. For separation of phospholipids, prepare a chloroform-methanol-H₂O (65:25:4, v/v/v) mixture (Knittelfelder and Kohlwein, 2017a and 2017b).
  2. For separation of neutral lipids, prepare a hexane-diethyl ether-acetic acid (70:30:1 v/v/v) mixture (Fuchs et al., 2015).
3. Add the mobile phase to glass TLC developing chambers. Saturate the chamber atmosphere with folded blotting paper for more than 30 min.
4. Activate the silica gel 60 plates at 100°C in an oven for 30 min. Draw a fine spotting line with a pencil 2 cm from the bottom of the TLC plate.
5. Spot 5-20 μl dissolved samples on the activated silica gel plates carefully with 10-μl pipette tips. Keep the spacing between these spots larger than 0.5 cm. In addition, spot 0.2-1 μg lipid standards (DAG or PC) to distinguish the complex bands of each sample and to monitor the developing and staining steps.
6. Develop each plate in a solvent chamber until the solvent front reaches the top of the plate. To maintain a solvent-saturated atmosphere in the chamber, do not open the lid while the plate is developing.
7. Dry the plates in a fume hood for 20 min at room temperature.
8. Stain the dried plates with a 0.03% Coomassie Brillant Blue R-250 solution containing 20% methanol for 15 min, and subsequently de-stain in 20% methanol for 10 min (Nakamura and Handa, 1984).
9. Dry the plates under room temperature for 1 h and take photos by a gel imaging system (Figure 2).
10. Analyze the grayscale of each lipid band using ImageJ (Steps: Open an image file in ImageJ – go to Analyze – gels – Select First Line – Select Next Line – Wand (tracing) Tool – Plot Lanes).
  
  Figure 2. Examplary HPTLC separation of yeast lipids. A. Neutral lipids: the solvent system was hexane-diethyl ether-acetic acid (70:30:1 v/v/v). DAG, diacylglycerol; TAG, triacylglycerol; SE, steryl esters. B. Polar lipids: the solvent system was chloroform-methanol-H₂O (65:25:4, v/v/v). FA, fatty acids; NL, neutral lipids; PE, phosphatidylethanolamine; CL, cardiolipin; PC, phosphatidylcholine; PS, phosphatidylserine. X, unidentified. Lipids were stained with Coomassie Brillant Blue R-250.

Acknowledgments

The authors would like to thank Dr. Zhou Peng and Prof. Xiaoling Miao (School of Life Sciences and Biotechnology, Shanghai Jiao Tong University) for their kind advice regarding experimental procedures. This work was supported by the National Natural Science Foundation of China (grants 91957104, 31671431, and 91754110). This protocol is adapted from Li et al. (2020) and works cited therein.

Competing interests

The authors declare no competing financial interests

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