Successful fluorochrome incorporation was confirmed by native polyacrylamide gel electrophoresis generating a single band at about 600 kDa corresponding to toxin A protein dimer [29, 30] under non-denaturing
conditions, which exhibited fluorescence during illumination with UV light. Toxin A488 was also shown to induce morphological changes in Vero cells and Caco-2 cells identical to that seen for unlabelled toxin A (treated as per labelled toxin A without the addition of Lapatinib ic50 the label), confirming that labelling had not compromised receptor-binding ability. To confirm that fluorescence in flow cytometry was because of toxin A488 only, without any contribution from free label that may have either not been removed following the labelling procedure or become detached from the toxin during storage or binding studies, toxin A488 was preincubated with PCG-4-conjugated beads prior to incubation with Caco-2 cells. A complete loss of Caco-2 cell-associated fluorescence was seen after incubation with the toxin A-depleted preparation (Fig. 1), confirming that all fluorescence was toxin A specific. In initial studies, following incubation of PBMNCs with toxin A488 at 37 °C for up to 24 h, monocytes were distinguished from lymphocytes by their forward- and side-scatter characteristics. In contrast to toxin A488-exposed lymphocytes, toxin
A488-exposed monocytes showed significant fluorescence at all time points up to 5 h, with HSP90 a peak at 1 h (Fig. 2A). Drop
in monocyte-associated fluorescence from 1 h onwards after exposure to Ruxolitinib cell line toxin A488 was associated with loss of events in the monocyte gate (Fig. 2B). The fluorescence level of toxin A488-exposed lymphocytes remained low, with no significant change (compared with control lymphocytes) over the 24 h period of study (Fig. 2A). Thus, at 24 h, there was no significant difference in fluorescence between lymphocytes incubated (at 37 °C) in control medium, compared with those cultured with toxin A488. In contrast to monocytes, the number of events in the lymphocyte gate (in toxin A488-exposed PBMNCs) did not change significantly from cells exposed to control medium over the 24 h period of study (Fig. 2B). When studied after 48-h incubation at 37 °C, fluorescence of toxin A488-exposed lymphocytes was marginally, but significantly greater than lymphocytes cultured with control medium (5.35 versus 4.97; P < 0.01). By contrast, following incubation at 4 °C, the difference in fluorescence between toxin A488-exposed and control lymphocytes fell short of statistical significance (5.0 versus 4.85; P = 0.07). The ability of trypan blue to quench fluorescence of monocytes exposed to toxin A488 at 37 and 4 °C was subsequently investigated. Initial studies, using PBMNCs labelled with anti-CD45 antibody, followed by labelling with Alexa Fluor 488-conjugated anti-mouse antibody, showed that trypan blue quenched 87.27 (±4.7)% of cell surface–associated fluorescence.