© Under License of Creative Commons Attribution 3.0 License
Materials & Method
Human malignant melanoma cells (MeWo) were grown at37
C and 5% CO
in Dulbecco’s minimal essential mediumsupplemented with 10% fetal bovine serum (DMEM,Life Technologies, Carlsbad CA). The MeWo cell line wasestablished from a skin biopsy of malignant melanoma, andis the standard cell line used to propagate VZV.
Varicella zoster virus (VZV) obtained from a zoster vesicleand shown to be of the European strain was used before the20
passage  Since VZV is highly cell-associated,virus was propagated by co-cultivation of infected cells withuninfected cells at ratios ranging from 1:4 to 1:100 dependingon use. VZV stocks were prepared using infection ratios of1:100 and dose response experiments were performed atinfection ratios of 1:4. .
Treatment with honey
Commercially obtained pure clover and Manuka honey werediluted to a nal concentration ranging from 0% to 6% (wt/ vol) in culture medium and lter sterilized.
Cell viability assay
Cell viability in various honey concentrations was determinedby neutral red uptake assay.  Briey, after 3 days in cultureMeWo cells were incubated for 3 hrs with 40 ug/ml neutralred (Sigma, St. Louis, MO) in DMEM, xed in 0.5% formal-dehyde, 1% CaCl2 for 1 min, dissolved in 50% ethanol, 1%acetic acid for 5 min, and optical density at 560 nm deter-mined on triplicate samples.
MeWo cells infected with VZV and uninfected MeWo cellswere incubated in culture medium containing various honeyconcentrations. After plaque development (3 days post-in-fection) cultures were xed in 4% formaldehyde, permea-bilized for 10 minutes in methanol/acetone (50:50; v/v) andextensively washed with Tris-buffered saline (TBS; 20 mMTris-HCl, pH 8.0, 150 mM NaCl). VZV plaques were identiedby immunostaining. Briey, monolayers were blocked for 60minutes in 3% blocked for 60 minutes in 3% BSA in TBS,incubated for 60 minutes with primary antibody (rabbit anti-IE63: 1:1,000 dilution)  followed by 60 minutes incubationwith alkaline phosphatase conjugated secondary antibody(goat anti-rabbit IgG; 1:10,000 dilution; Abcam, Cambridge,MA). Immunostaining was visualized with NBT/BCIP (Pierce,Rockford IL). Between all incubations, the cultures underwentextensive washing in TBS. Plaques were counted with the aidof a dissecting microscope (4 x magnications).
Results & Discussion
Both clover and Manuka honey are readily soluble in water,and at low concentrations inhibit neutral red uptake to equalamounts. MeWo cells in both honey concentrations < 3.75%showed equal survival slopes (
). This is most likely due to
Viability of MeWo Cells in dilutehoney solutions.Neutral red uptake assay was usedto determine cell viability in varioushoney concentrations. MeWo cellswere incubated for 3 hrs with 40 ug/ ml neutral red in DMEM, xed in 0.5%formaldehyde, 1% CaCl2 for 1 min,dissolved in 50% ethanol, 1% acetic acidfor 5 min. Optical density was determinedat 560 nm on triplicate samples.Cytotoxicity for both honeys is similaruntil highest concentration tested. At 5% dilution, clover honey is more toxic toMeWo cells than Manuka honey.
% survival of MeWoas a function of honey concentration
% honey (wt/vol)
% c e l l s u r v i v a l ( n e u t r a l r e d a s s a y )