1% Triton X 100 for 15 min and incubated in 5% FCS 0. 1% Tween PBS for 1 h. Cel

The fluorescence inten sity was measured using Glomax spectrofluorometer at wavelengths on excitation and emis sion buy INNO-406 400 nm and 505 nm, respectively. Plasma membrane permeability assay The loss of plasma membrane integrity was analyzed using of fluorescent DNA binding dye 7 AAD, EL 4 cells were washed once in PBS and resuspended in 0. 5 ml of staining solution, 7 AAD fluorescence of cells was analyzed by flow cytometry using FL3 channel. In each sample at least 5,000 events were collected. The data was analyzed using FlowJo and WinMDI software. Assessment of mitochondrial membrane potential in living cells Mitochondrial membrane potential was mea sured using fluorescent dye 3,3 dihexyloxacarbocyanine iodide and 5,5,6,6 tetrachloro 1,1,3,3 tet raethylbenzimi dazolylcarbocyanine iodide, The cell suspension was adjusted to a density of 1 × 106 cell ml and incubated in complete medium for 15 min at RT in the dark with 20 nM DiOC6 or with 2 ug ml JC 1.

After which, buy Lapatinib the cells were washed twice in cold PBS, sus pended in a total volume of 500 ul and analyzed by flow cytometry, or FL1 and FL2 channels for JC 1. In each sample at least 5,000events were collected. The data was analyzed using FlowJo and WinMDI software. ELISA Polystyrene microtiter plates were coated with gangliosides GD2, GM2, GD1b and GD3 that were obtained according to the method applied in our previous work, or kindly provided by Dr. Mikhalyov at concentration 0. 25 ug in 100 ul of 70% methanol per well. Following air drying, all wells of the plate were blocked with 2% BSA in PBS T in 100 ul per well for 2 h at RT.

Antibodies were added in triplicates Lonafarnib 構造 at different concentrations. Following incubation for 2 h at 37 C and washing with PBS T, HRP goat anti mouse IgG were added. After incubation for 1 h at 37 C and further washing, TMB color reaction was performed and OD was read using Multiscan FC microplate reader at 450 nm. Percent of cross reactivity was measured as ratio of OD450 of TMB substrate in GM2, GD1b or GD3 coated wells to OD450 of TMB substrate in GD2 coated wells. The amount of gangliosides adsorbed to each well was determined by using fluorescent labeled gangliosides BODIPY FL C5 GM1 and BODIPY FL C5 GD3, Fluorescent probes were coated at the same concentration as unlabeled ganglio sides, and the same operations were performed for fluorescent probes except adding of antibodies.

At the last stage BODIPY labeled gangliosides GM1 and GD3 that were adsorbed on surfaces of the wells were subsequently dissolved in methanol and fluorescence was measured using a Dynatech Micro FLUOR Reader, The amount of ganglio sides that were adsorbed on the wells was measured using proper calibration curve, RFU BODIPY FL C5 GM1 36. 396 , All experiments were repeated three times. Modulation of GD2 expression Downregulation of GD2 expression using PDMP inhibitor In the initial experiments we determined optimal con centration of PDMP inhibitor and time of incubation to downregulate GD2 expression in EL 4 cells. EL 4 cells were treated with different concentrations of PDMP for 2 7 days. The expression of GD2, cell viability, and cell death were analyzed by flow cy tometry using surface staining for GD2, PI, and MTT tests.