Brown-yellow granules Sotrastaurin concentration that appeared in the cells indicated a Selleckchem Poziotinib positive result . In vitro immunofluorescence MGC80-3 cells and GES-1 at a concentration of 5 × 104 cells/ml were seeded separately onto four 35-mm culture dishes with glass bottoms (1 ml in each dish). The four 35-mm culture dishes of MGC80-3 were marked A, B, C, and D, while those of the GES-1 group were marked E, F, G, and H. After 24 h of culture, the cells were washed with PBS twice. The experimental dishes B and F were added with 100 μl of CC49-QDs Ab probe (337.5 nmol). The negative control dishes A and E were added 100 μl of QDs (337.5
nmol) for the purpose of insteading of the CC49-QDs Ab probe. The cells in the four dishes described above were incubated for 1 h at 37°C and then washed with PBS three times. The competitive group dishes C and G were added to 200 μl of CC49 monoclonal antibody (1 μg/ml) for 2 h of blocking. Subsequently, the cells were washed Selleckchem R428 with PBS twice, and then an equimolar amount of CC49-QDs Ab probe was added to the experimental dishes. To the positive control dishes D and H, 100 μl of CC49 monoclonal antibody (1 μg/ml) was added for 2 h of blocking. After washing three times (each for 3 min), fluorescent
secondary antibody (goat against mouse IgG and conjugated to fluorescein isothiocyanate, 1:100) was added for another 30 min of incubation. 4′,6-Diamidino-2-phenylindole (DAPI) was used to label the cell nucleus before imaging with a fluorescence microscope. In the fluorescence imaging of the cancer cells, the cell nucleus stained with DAPI (A1/B1/C1/D1 in Figure 1 and E1/F1/G1/H1 in Figure 2) was observed under the UV mode in which the excitation wavelength was 330 to 380 nm and the emission wavelength was 400 to
420 selleck kinase inhibitor nm. MGC80-3 cells labeled with QDs (A2 in Figure 1) and CC49-QDS (B2 and C2 in Figure 1) were observed under the G-2A mode in which the excitation wavelength was 510 to 560 nm and the emission wavelength was 575 to 590 nm. GES-1 cells labeled with QDs (E2 in Figure 2) and CC49-QDS (F2 and G2 in Figure 2) were observed under the same mode. MGC80-3 cells (D2 in Figure 1) and GES-1 cells (H2 in Figure 2) labeled with fluorescent secondary antibody were imaged under the FITC mode in which the excitation wavelength was 465 to 490 nm and the emission wavelength was 505 to 520 nm. All the experiments were repeated three times. Figure 1 In vitro labeling of MGC80-3 cells with CC49-QDs Ab probe and primary QDs. (A1/B1/C1/D1) The cell nucleus was stained with DAPI. (A2) MGC80-3 cells labeled with QDs. (B2) MGC80-3 cells labeled with CC49-QDs. (C2) MGC80-3 cells labeled with CC49-QDs after blocked with free CC49. (D2) MGC80-3 cells labeled with fluorescent secondary antibody. A3/B3/C3/D3 were merged with A1 and A2, B1 and B2, C1 and C2, D1 and D2, respectively.