In this study, we have labeled L6 cells with IgG conjugated QDs in 4 ��C. The experimental time was reduced by using QD-IgG conjugates. The GLUT4-QD complex was located on PM and internalization of GLUT4 selleck chem inhibitor can be observed after this simplified labeling procedure. Coupled with Andor Revolution XD laser confocal microscope system, three dimensional trajectory of GLUT4 in live L6 cells can be observed clearly in real time.2.?Materials and Methods2.1. MaterialsAnti-c-myc monoclonal antibody 9E10 and Qdot 605 goat anti-mouse IgG conjugate were purchased from Invitrogen. L6-GLUT4myc Inhibitors,Modulators,Libraries cells were provided by Prof. Amira Klip, the Hospital for Sick Children (Toronto, Ontario, Canada). GLUT4-EGFP plasmid was provided by Prof. Tao Xu (Institute of Biophysics of the Chinese Academy of Sciences, Beijing, China).

Other chemicals were purchased from Invitrogen.2.2. Cell Inhibitors,Modulators,Libraries CultureL6-GLUT4myc myoblasts were cultured in ��-MEM supplemented with 10% fetal bovine serum and 1% antibiotic at 37 with 5% CO2.2.3. Labeling GLUT4myc Molecular with QDsL6-GLUT4myc cells were seeded in growth medium (��-MEM, 10% fetal bovine serum, and 1% antibiotic) at 30�C40% confluence the previous day. They were starved with depletion medium (��-MEM and Inhibitors,Modulators,Libraries 1% antibiotic) for 3 h at 37 ��C prior to experimentation to enhance the effect of insulin stimulation. Then, they were stimulated with 100 nM insulin at 37 ��C for 20 min. Later, they were blocked with 5% goat serum in ��-MEM containing 100 nM insulin for 10 min at 37 ��C and then incubated with anti-myc monoclonal antibody 9E10 (with an initial concentration of 0.

5 mg/mL, 1:100 diluted in ��-MEM) containing 100 nM insulin for 1 h at 37 ��C. Unbound antibody was aspirated away and the cover slips were washed three times with ice-cold ��-MEM containing 100 nM insulin at 4 ��C. Later, they were blocked with 5% goat serum in ��-MEM containing 100 nM Inhibitors,Modulators,Libraries insulin for 10 min at 4 ��C and incubated with Qdot 605 goat anti-mouse IgG conjugate Batimastat (with an initial concentration of 1 ��M, 1:50 diluted in ��-MEM) containing 100 nM insulin for 1 h at 4 ��C. Unbound Qdot-IgG was aspirated away and the cover slips were washed five times with ice-cold ��-MEM containing 100 nM insulin at 4 ��C. Finally, the cells were cultured in growth medium at 37 ��C for imaging. For control, two groups were prepared.

One group was stimulated with insulin and then incubated research use only with QD-IgG diluted in growth medium containing 100 nM insulin for 1 h at 4 ��C, the primary antibody 9E10 was omitted during the labeling procedure in this group. Another group was not stimulated with insulin and insulin was absent throughout the labeling procedure.2.4. Transfection with GLUT4-EGFPL6-GLUT4myc cells were seeded in growth medium (��-MEM, 10% fetal bovine serum, and 1% antibiotic) at 80% confluence prior to experimentation. For each chamber, 0.8 ��g GLUT4-EGFP plasmid was diluted into 50 ��L OMEM without fetal bovine serum.