Every time before cells are sorted by fluorescence intensity, all cells are gated by FSC (forward-scatter) and SSC (side-scatter) (this means that all cells are selected by size and granule intensity) (Fig. IA, IIIA) to prevent dead or aggregated cells from being analyzed. Then, using a portion of the selected cells, we start FACS analysis and determine which cells should be further selected by fluorescence intensity (Fig. IB, IIIB). After several rounds of this sorting procedure, the selected cells are accumulated as shown in Fig. IIB and Fig. IVA.
When we tried to clone adiponectin receptors, we first attempted to identify adiponectin receptor-positive cells by determining binding to globular adiponectin conjugated with the red fluorescent probe. Fig. IIA showed cells that were sorted with the high intensity of red fluorescence (FL1). However, we could not find adiponectin receptor-positive cells after all this procedure. As adiponectin has been reported to be physically of sticky nature, we thought that this high intensity of red fluorescence might have been brought about by a non-specific binding of adiponectin. Then, we further incubated these red cells, which had been selected by red fluorescence, with globular adiponectin conjugated with a green fluorescent probe. Nonspecific binding is usually determined using an excess of unlabeled or differently labeled adiponectin. Specific binding is determined by subtracting the nonspecific binding from the total binding. In this case, by this incubation (first incubated with globular adiponectin conjugated with a red fluorescent probe, then incubated with an excess of globular adiponectin conjugated with a green fluorescent probe), we expected that the cells possessing specific binding sites for globular adiponectin might change its color from red to green, which would lead to identification of adiponectin receptor-positive cells. Because of the necessity for this dual color scanning by red and green fluorescent probes, the X-Y axes of Fig. IIA needed to be exchanged (namely Fig. IIA was converted to Fig. IIB). Then we did FACS analysis and determined the cells to be selected by both the high intensity of green fluorescence (FL1) and the low intensity of red fluorescence (FL2) (Fig. IIIB). After several rounds of sorting these cells, the finally selected cells were accumulated as shown in Fig. IVA and from these cells we successfully cloned adiponectin receptors.