THP-1 and HEK293 cells are transiently transfected using DEAE-dextran and Polyfect reagent, respectively. Plasmids transfected contain genes coding for the NF-κB-dependent pELAM-luc luciferase reporter, TLR2, TLR4, CD14, and MD2. Cells (5×10⁵ THP-1 or 1×10⁵ HEK293) are added to 12-well plates, washed after 18 h, and stimulated for 5 h. Cells are then lysed with reporter lysis buffer as directed, and lysates are analyzed for luminescence using Promega luciferase substrate and a Monolight 3010 luminometer.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

The kinetics of cell death induced by AmB against Leishmania promastigotes is followed by using fluorometry with the DNA-binding compound ethidium bromide (EB). Fluorescence measurements are performed on a SPEX Fluorolog II spectrophotometer at 365-580 nm excitation-emission wavelengths. Promastigotes at a final concentration of 25×10⁶ cells/mL are incubated for 5 min with gentle stirring in the fluorescence cuvette with 2 mL of different buffered solutions but always containing 10 mM glucose and EB (50 mM). After signal stabilization is achieved, AmB is added and dissolved in dimethylsulfoxide. Maximal EB incorporation is always obtained by adding digitonin (50 mg/mL). All solutions used are buffered with 75 mM TRIS (pH 4 7.6) and contain 150 mM NaCl (BNa⁺), 150 mM KCl (BK⁺), 150 mM choline chloride, and 100 mM sucrose, 100 mM NaCl. The osmolarity of all solutions is always adjusted to 390±5 mOsm using an advanced instrument SW2 osmometer.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Sau K, et al. The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism. J Biol Chem. 2003 Sep 26;278(39):37561-8. Epub 2003 Jul 14.

  [2]. Barwicz J, et al. The effect of aggregation state of amphotericin-B on its interactions with cholesterol- or ergosterol-containing phosphatidylcholine monolayers. Chem Phys Lipids. 1997 Feb 28;85(2):145-55.

  [3]. Ramos H, et al. Amphotericin B kills unicellular leishmanias by forming aqueous pores permeable to small cations and anions. J Membr Biol. 1996 Jul;152(1):65-75.

  [4]. Demaimay R, et al. Pharmacological studies of a new derivative of amphotericin B, MS-8209, in mouse and hamster scrapie. J Gen Virol. 1994 Sep;75 (Pt 9):2499-503.

  [5]. Adams ML, et al. Amphotericin B encapsulated in micelles based on poly(ethylene oxide)-block-poly(L-amino acid) derivatives exerts reduced in vitro hemolysis but maintains potent in vivo antifungal activity. Biomacromolecules. 2003 May-Jun;4(3):750-7.

924.08

C₄₇H₇₃NO₁₇

1397-89-3

C[C@H]([C@@H](O)[C@@H](C)[C@H](C)OC1=O)/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](O[C@]2([H])O[C@H](C)[C@@H](O)[C@H](N)[C@@H]2O)C[C@@]3([H])[C@H](C(O)=O)[C@@H](O)C[C@](C[C@@H](O)C[C@@H](O)[C@H](O)CC[C@@H](O)C[C@@H](O)C1)(O)O3

4°C, protect from light, stored under nitrogen

Room temperature in continental US; may vary elsewhere