On the other hand, biotinylated purified Bt 18 toxin tagged with FITC-conjugated streptavidin appeared green or greenish yellow (as a result of see more overlap with Alexa Fluor® 594). For CEM-SS (Figure 5), biotinylated purified Bt 18 toxin appeared at all test intervals (1, 2, 12 and 24 hours). The intensity and extent of staining increased relatively with increased incubation period. The biotinylated toxin was seen at the periphery of

the cell where the cell membrane was located. For human T lymphocytes (Figure 6), biotinylated purified Bt 18 toxin did not appear at all test intervals except at 24 hours. Compared to CEM-SS at the same Adavosertib interval, the intensity and extent of staining was very much less remarkable. Figure 5 Confocal microscopic appearance of biotinylated purified Bt 18 toxin-treated CEM-SS. CEM-SS cells were treated with fixed concentration of biotinylated purified Bt 18 toxin at various time intervals. The biotinylated toxin was then tagged

with FITC-conjugated streptavidin (green). The biotinylated toxin was found at the periphery of treated CEM-SS cells. Increased binding of the biotinylated toxin was observed with increased incubation period. (A) Untreated negative control at 24 hours. (B) to (E) Treated cells at 1, 2, 12 and 24 hours respectively. Magnification: 630×. B = Biotinylated purified Bt 18 toxin, M = Cell membrane, N = Nucleus. Selleck GDC0068 ID-8 Bar = 10 μm. Figure 6 Confocal microscopic appearance of biotinylated purified Bt 18 toxin-treated human T lymphocytes. Human T lymphocytes were treated with fixed concentration of biotinylated purified Bt 18 toxin at various time intervals. The biotinylated toxin was then tagged with FITC-conjugated streptavidin (green). Biotinylated toxin was not found at all intervals except 24 hours, where the binding was very minimal. (A) Untreated negative control at 24 hours. (B) to (E) Treated cells at 1, 2, 12 and 24 hours respectively. Magnification:

630 ×. B = Biotinylated purified Bt 18 toxin, M = Cell membrane, N = Nucleus. Bar = 10 μm. Discussion Binding studies related to Bt have largely been carried out in insects mainly for characterisation of Bt toxins and the determination of Bt toxin resistance in insects [14]. According to data from cell viability assays in this study, biotinylation did not affect the biological activity of the toxin. This phenomenon was observed in other studies [15, 16]. Results from the homologous competitive assays suggested that purified Bt 18 toxin had a higher affinity for CEM-SS, followed by CCRF-SB and CCRF-HSB-2 as the dissociation constant is inversely proportional to the binding affinity. For MCF-7, the dissociation constant could not be obtained because the inhibitory concentration (IC50) was not achieved.