Melittin

Cell experiment:

Melittin is purified from bee venom by reversed phase liquid chromatography and reconstituted in sterile water to form a stock solution of 1 mg/mL before storage at -20 °C until required for analysis. Cell viability is assessed by an Alamar Blue (AB) cell viability reagent. Both A2780 and A2780CR cells are seeded at 1×104 cells/well in 96-well plates and incubated at 37 °C and 5% CO2 in a humidified atmosphere for 24 h. After this incubation period, the cells are treated with various concentrations of Melittin ranging from 0.5 to 14 µg/mL in 100 μL of medium, and re-incubated at 37 °C and 5% CO2 for a further 24 h. Triton X at 1% (v/v) and cell culture media are used as positive and negative controls, respectively. After this, AB is added at a final concentration of 10% (v/v) and the resultant mixture is incubated for a further 4 h at 37 °C and 5% CO2. Then, the plates are read at an excitation wavelength of 560 nm and the emission at 590 nm is recorded on a SpectraMax M3 microplate reader . Background-corrected fluorescence readings are converted to cell viability data for each test well by expressing them as percentages relative to the mean negative control value[2].

References:

[1]. Steiner MR, et al. Responses of purified phospholipases A2 to phospholipase A2 activating protein (PLAP) and Melittin. Biochim Biophys Acta. 1993 Feb 10;1166(1):124-30.
[2]. Alonezi S, et al. Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology. Metabolites. 2016 Oct 13;6(4). pii: E35.