The apparent dissociation constant was measured by incubating ~106 Hep3B cells with different concentrations of IRDye800 labeled peptide ranging between 0–200 nM at 25°C for 1 hour, and washed with cold PBS. The mean fluorescence intensities were measured with flow cytometry (BD LSR Fortessa, BD Biosciences). The equilibrium dissociation constant kd = 1/ka was calculated by performing a least squares fit of the data to the non-linear equation I = (I0+Imaxka[X])/(I0+ka[X]).58 (link) I0 and Imax are the initial and maximum fluorescence intensities, corresponding to no peptide and at saturation, respectively. [X] represents the concentration of bound peptides. Graphpad Prism ver 8 software was used to plot and fit the data.
The apparent association time constant k was measured by incubating ~106 Hep3B cells with 5 μM of IRDye800-labeled peptides at time intervals between 0–40 min at 25°C. The cells were centrifuged and washed with cold PBS. Flow cytometry was performed using Sony iCyt SY3200 at λem = 775/50 nm with λex = 685 nm excitation, and the median fluorescence intensity (y) was ratioed with that found without peptide at different time points (t) using Flowjo (ver 10.1r5) software. The rate constant k was calculated by fitting the data to a first order kinetics model, y(t) = Imax[1-exp(−kt)], where Imax = maximum value.59 (link)
The apparent association time constant k was measured by incubating ~106 Hep3B cells with 5 μM of IRDye800-labeled peptides at time intervals between 0–40 min at 25°C. The cells were centrifuged and washed with cold PBS. Flow cytometry was performed using Sony iCyt SY3200 at λem = 775/50 nm with λex = 685 nm excitation, and the median fluorescence intensity (y) was ratioed with that found without peptide at different time points (t) using Flowjo (ver 10.1r5) software. The rate constant k was calculated by fitting the data to a first order kinetics model, y(t) = Imax[1-exp(−kt)], where Imax = maximum value.59 (link)
