Dicetyl phosphate

Letrozole was kindly donated by Tofigh Daru (Tehran, Iran). Sorbitan monolaurate (Span20, Cat# S6635), Sorbitan monostearate (Span60, Cat# S7710), Sorbitan monooleate (Span80, Cat# S6760), Cholesterol (CHOL, Cat# C8667), dicetylphosphate (DCP, Cat# 850042P), Sodium dodecyl sulfate (SDS, Cat# L3771) and dialysis membrane (MWCO 12kDa, Cat# D6191), Nile red and Coumarin 6 were obtained from Sigma Aldrich (USA). Dimethyl sulfoxide (DMSO, Cat# 109678), chloroform (Cat# 107024), Amicon (Ultra-15-Membrane, MWCO 30kDa, Cat# C82301) were purchased from Merck (Germany). Trypsin-EDTA (Cat# 15400054), Trypan blue (Cat# 15250061), RPMI-1640 culture medium, DMEM (Dulbecco’s Modified Eagle culture medium, Cat# 11965092), phosphate-buffered saline (PBS, Cat# 10010049), fetal bovine serum (FBS, Cat# 16000044), MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (Cat# M6494) and penicillin/streptomycin (PS, Cat# 10378016) 100 X were purchased from Gibco (USA). The MDA-MB-231, MCF-7, and MCF10A cell lines were obtained from Pasteur Cell Bank (Iran). The Annexin V-FITC flow cytometric kit was obtained from Affymetrix Biosciences (USA). RNA extraction and cDNA synthesis kits were purchased from Transgene Biotech, China (Cat#. ER101-01 and AE301-02).

Bovine serum albumin (BSA), cholesterol, curcumin, dicetyl phosphate (DCP), 2,6-dichloroindophenol (DCIP), didodecyldimethylammonium bromide (DMAB), phosphatidylethanolamine (PE), poly(d,l-lactide-co-glycolide) (PLGA) (Resomer RG 85:15H), phenazine methosulfate (PMS), and succinic acid were purchased from Sigma-Aldrich (St Louis, MO, USA). CM-H₂DCFDA was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ethyl acetate (AR grade) was procured from Rankem Fine Chemicals (New Delhi, India). Chloroform and methanol were purchased from E Merck (Darmstadt, Germany). All other reagents were of analytical grade.

Phosphatidylethanolamine (PE), cholesterol, dicetyl phosphate (DCP), stearylamine, poly (D,L-lactide-co-glycolide) (PLGA) (molecular weight: 50,000–75,000; lactide:glycolide 85:15), and didodecyl dimethyl ammonium bromide (DDAB), bovine serum albumin (BSA), 2,6-dichloroindophenol (DCIP), phenazine methosulfate (PMS), and succinic acid were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ethyl acetate (EA; AR grade) was obtained from Rankem Fine Chemicals (New Delhi, India). Methanol and chloroform were obtained from E Merck (Darmstadt, Germany). Both melatonin and NaF were obtained from MP Bio medicals, LLC (Santa Ana, CA, USA). Other chemicals used were of analytical grade.

Mice were injected with 50 μg of recombinant TgO/GABA-AT, subcutaneously. The protein was formulated with an NISV (nonionic surfactant vesicle) preparation to act as an adjuvant. The vesicles were made by melting mono-palmitoyl glycerol, cholesterol, and dicetyl-phosphate (All from Sigma, UK) in a molar ratio of 5:4:1. Vesicles formed following the addition of 37°C PBS pH 7.4 and vigorously vortexed for 2 min. Vesicle preparations were lyophilized and subsequently rehydrated with the appropriate protein dilutions. These preparations were stored at −20°C until use and heated to 37°C just prior to injection. Mice were given 2 injections of the preparations, 2 weeks apart. Ten days after the final injection, the mice were euthanized by CO₂ inhalation, and blood was obtained by cardiac puncture. Blood samples were centrifuged at 13,000 rpm for 10 min at 4°C. Serum was collected and stored at −20°C prior to testing.