Fluorescent chemokines, monitoring

Monitoring Scavenging with Radiolabeled Chemokines Monitoring Scavenging with Fluorescent Chemokines... 

Measure the fluorescence emission at 505 nm using 340 nm excitation. Monitor the baseline. When a stable baseline is obtained for 10-30 s, add the chemokine to be tested. After the peak of calcium mobilization has subsided (about 1 min), calibrate the assay by adding 20 pL of minimum buffer, allow for stabilization, then add 20 pL of maximum buffer to dissolve cell membranes in order to obtain full complexation of fura-2 with Ca2+. Record the baseline, the peak, the minimum and the maximum values (see Fig. 1). The concentration of Ca2+ mobilized is calculated according to the following modified formula (6) ... 

We first describe two methods to generate fluorescent-labeled chemokines. Part A describes the expression and purification of chemokines fused to fluorescent proteins and expressed in insect cells. Part B delineates the expression of chemokines in bacteria, folding, and purification, followed by enzymatic labeHng with phosphopantetheinyl transferases. In the second section, we provide protocols to monitor chemokine uptake by microscopy and flow cytometry (FACS). 

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