E Change in the ABCG2 expression at the protein STAT Signaling Pathway level, flow cytometry of ABCG2 expression in the contr’and the treated cells was determined using the method of Minderman et al. with slight modifications. Briefly, cells with IL-1b, IL-6 or TNF were pretreated for 72 h. Then 0.51 9106 cells were washed with PBS and fixed in 3.7% formaldehyde for 10 min at room temperature and permeabilized in ice-cold 90% methanol for 10 min. Subsequently End the cells were washed with PBST and incubated with 10% BSA for 30 min at room temperature. The cells were then incubated for 60 min on ice with a primary Ren monoclonal BXP body 21 which Recogn t internal epitopes of the protein ABCG2. The cells were washed with PBST and incubated on ice for 20 min with fluorescein isothiocyanate-conjugated goat anti-mouse antibody Body to the prime Ren anti-ABCG2 monoclonal antibody capture. After the last wash, the cells were resuspended in PBST and kept on ice in the dark prior to analysis. FITC fluorescence was measured on a FACSCalibur flow cytometer using an argon laser excitation at 488 nm measured standard equipped, and with bandpass 530/30 p38 MAPK Signaling Pathway filter FL1. Not lebensf Hige cells are on the gate-scattering at the front and side, and analyzes a total of 1.0 9104 cells.
The intensity of t, the background fluorescence by a bcl-2 control sample of cells in the absence of primary Rated r Mab incubated. Flow cytometry data were processed and analyzed with WinMDI software version 2.8. All tests were conducted in at least three independent Ngigen performed experiments. Relative expression of ABCG2 was treated as the ratio Ratio of MFI histogram for each of the samples in the MFI of the untreated sample can be divided into k. Studies using functional efflux assay for efflux and modulation of the carrier Gers to investigate the dosage of cytokines were trypsinized and the cells washed with PBS. In the accumulation phase, the cells were incubated in RPMI 1640 with 10% FCS with 3 MX LM with or without 10 IN fumitremorgin C as a modulator at 37 for 30 min. The cells were then immediately transferred on ice, washed twice with ice-cold PBS and resuspended in 1 ml completely Ndigem Voriconazole medium with or without free MX 10 lm FTC.
Then, the incubation for 1 h at 37 was continued in order to align maximum efflux of MX to erm. The cells were washed once after the efflux, and conclude Lich resuspended in 1 ml of ice-cold PBS. The cells were then kept on ice in the dark until analysis. The intracellular Re MX fluorescence was measured h in a flow cytometry using a FACSCalibur flow cytometer with an argon laser at 488 nm and the standard of driving a bandpass filter at 650 nm. The efflux histogram of cells incubated for 30 min with MX and then lie one transport for 1 h efflux in complete medium alone was produced, was the histogram FTC / Ausflu cells were incubated from 30 min with MX and FTC, by incubation in complete medium and the FTC followed for 1 h. The modulating effects of proinflammatory cytokines on the MX-transporter in the cells was assessed by the MFI of the efflux histograms. % Change in MFI mediation FTC / 9100: nally ABCG2 efflux was in the presence and absence of FTC by calculating the relative increase in MFI compared as follows. Statistics Results are expressed as mean ± SD of three independent Ngigen experiments, and the importance is to determine.
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