Total lipids from mycobacteria were prepared as described previously89. Birefly, bacterial cells were extracted at 100 mg/ml in chloroform:methanol (C/M) (2:1, v/v) for 1 h at room temperature. After the centrifugation, the supernatant was filtered and evaporated in vacuo to weigh the recovered amounts of the total lipids. GMM and GroMM were prepared as described previously7,16,90. For GMM preparation, the total lipids dissolved in C/M (2:1) were added with 20 volumes of ice-cold acetone and incubated for 30 min on ice. After centrifugation, the pellet was washed with ice-cold acetone, dissolved with C/M (2:1), and fractionated by TLC (Analtech #Z26551) with chloroform/methanol/acetone/acetic acid (90:10:10:1, v/v), followed by fractionated with chloroform/acetone/methanol/water (50:60:2.5:0.6, v/v). The spot corresponding to GMM was scraped off the silica gel plate and extracted with C/M (2:1), dried, and rinsed several times with methanol at room temperature to remove residual contamination of glycopeptidolipids and phospholipids. For GroMM preparation, the total lipids extracted from the bacteria cultured in 7H10 medium containing 10% of glycerol were fractioned by two cycle of TLC with chloroform/ethyl acetone (5:1, v/v). The lipid spots were visualized with iodine vapor, and the spot corresponding to GroMM was scraped off the silica gel plate, followed by elution with C/M (2:1). fMAs were isolated from M. bovis BCG (Tokyo 172 strain, Japan BCG laboratory). The BCG cells were suspended in 85% tetrahydrofuran (THF)/water solution under a nitrogen atmosphere, followed by reflux with stirring for 1 h. The cell suspension was filtrated under pressure and washed with 75% THF/water solution. The residue was resuspended in 75% THF/water solution under a nitrogen atmosphere, followed by reflux with stirring for 1 h. The suspension was filtrated under pressure and washed with 75% THF/water solution three times and with methanol twice. Then, the bacterial cells were suspended in 50% 2-propanol/water solution containing 10% potassium hydroxide, followed by reflux with stirring for 2 h to complete alkaline hydrolysis of MA ester. After the refluxing, the suspension was cooled down on ice, and acidified with 6 M hydrochloric acid. The reaction mixture was extracted twice with n-heptane, and the n-heptane fraction was washed twice with water and then twice with 90% ethanol/water. Finally, the n-heptane fraction was concentrated in vacuo to obtain purified MAs. The final preparations of GMM, GroMM, and fMA were applied for TLC to confirm no extra no spots and for MADI-TOF mass spectrometry analyses to confirm the identity of the lipids.

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