Propylene glycol

Lipid droplets in mature adipocytes can be identified using Oil-Red-O staining solution. After fixing with 10% buffered formalin phosphate (Fisher Scientific, cat# SF100-4), cells were incubated with 100% propylene glycol (Amresco, cat# 0575) for 5 min at room temperature, followed by its removal and staining with Whatman filter paper-filtered Oil Red O solution (Electron Microscopy Sciences, cat# 36609-01) for 30 min to 2 h with gentle rocking. Next, staining solution was removed and cells were incubated with 85% propylene glycol (Fisher Scientific, cat# A426P) for 5 min, followed by rinsing in distilled water three times. Stained cells would remain covered in distilled water, parafilmed and stored at 4 °C refrigerator. Whole-well images were taken using a Leica dissection microscope. For Oil-Red-O staining quantification, cells samples were air dried overnight after removing water and Oil-Red-O stain was extracted using 150 μl/well (24-well plate) 100% isopropyl alcohol for 1 min. Optical density readings for extracted stain solution were measured at 510 and 690 nm in an ELx800 96 well plate reader (BioTek).

SZ95 sebocytes were cultured on coverslips in the presence of TLR1/2 and 4 ligands (PAM3CSK4 and LPS, respectively), arachidonic acid or vehicle for 24 and 48 hours as described previously. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min. Coverslips were placed in 100% propylene glycol (Amresco, Solon, OH, USA) for 1 min, followed by a rinse with distilled water. Cells were stained with 0.7% Oil Red O (Sigma-Aldrich) solution for 7 minutes then rinsed with 85% propylene glycol solutions. Nuclei were counterstained with hematoxylin and slides were covered using Mount Quick Aqueous mounting medium (Bio Optica, Milano, Italy).

Certified cannabinoid reference standards (at 1 mg/mL in methanol) including cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), CBD, tetrahydrocannabivarin, cannabinol (CBN), Δ⁹-THC, cannabichromene (CBC), cannabidivarin (CBDV), and Δ⁹-tetrahydrocannabinolic acid-A (THCA) were purchased from Cerilliant (Round Rock, TX). Second-source standards of CBN, CBD, and Δ⁹-THC were from Restek (Bellefonte, PA). CBD hemp oil was purchased from HempMeds (Poway, CA). Norgestrel, 4-pentylphenyl 4-methylbenzoate (PPMB), ammonium formate, formic acid, and sesame oil were from Sigma-Aldrich (St. Louis, MO). Medium-chain triglyceride (MCT) oil was from Warner Graham (Cockeysville, MD). Propylene glycol was from J.T. Baker (Central Valley, PA). Ammonium formate, formic acid, methanol, acetonitrile, and water were HPLC grade. All other reagents used were analytical grade. Bulk cannabis plant material was obtained from the National Institute on Drug Abuse (NIDA), National Institutes of Health, Bethesda, MD.

D-limonene (97%) were purchased from Alfa Aesar (Tewksbury, MA USA). Plate count agar and Tryptone soy broth were purchased from Oxoid (Basingstoke, Hampshire, United Kingdom). Ethanol (95%) was purchased from Echo Chemical Co. (Miaoli County, Taiwan). Acetone were purchased from Seedchem (Box Hill, VIC, Australia). Sulfuric acid was purchased from Aencore Chemical Co. (Box Hill, VIC, Australia). Sodium chloride, disodium hydrogen phosphate, sodium hydroxide, and sodium tetraborate were purchased from Showa Chemical Industry Co. (Minato-ku, Tokyo, Japan). Potassium dihydrogen phosphate were purchased from Katayama Chemical Industries Co. (Hyogo, Japan). Propylene glycol were purchased from JT Baker (Phillipsburg, NJ, USA). Tween 80, glucose, phenol, and pectin (from citrus peel) were purchased from Sigma Aldrich (Quentin Fallavier, France). The 3-Phenylphenol and sodium alginate were purchased from Acros organics (Trenton, NJ, USA). Potassium hydrogen phthalate was purchased from Fullin Nihon Shiyaku (Taoyuan, Taiwan). Dichloran rose Bengal chloramphenicol agar was purchased from Becton, Dickinson and Co. (Washington, DC, USA).

Ciclopirox olamine (CPO) batch 19D11-B023-0486 was purchased from Fagron (Rotterdam, The Netherlands). Micolamina^® lacquer batch T266 (CPO 8%, isopropyl alcohol, ethyl acetate, polymeth-acryliccopolyethylacrylate and dimethylsulfoxide) was purchased from a local distributor in Recife, Brazil. Isopropyl myristate, polyethylene glycol 400, Tween^® 20 (Polyoxy-ethylene sorbitan monolaurate), Span^® 80 (Sorbitan monooleate), Tween^® 80 (Polyox-yethylene sorbitan monooleate), ethyl alcohol, Kolliphor^® EL (Macrogolglycerol ricinoleate), Poloxamer 407, propylene glycol, isopropyl alcohol, potassium hydroxide, urea, hydroxypropylmethylcellulose (HPMC), sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium azide, oleic acid, BRIJ^®20 (Polyoxy-ethylene 20 oleyl ether), sodium lauryl sulfate (SLS), and HPLC solvents methanol and acetonitrile from JT Baker, Phillipsburg, NJ, USA, were purchased from Casa do laboratório, Pernambuco, Brazil. Sabouraud dextrose agar and mycosel agar (PL1340) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Plastlabor (Rio de Janeiro, Brazil), respectively.