Figure 4.
Stability of Radioligand 125I-Tyr-flg22 in Cell Suspension at Room Temperature.
HPLC analysis of free radioligand and radioligand bound to cells was reproduced for four time points (between 1 and 120 min of incubation) in two independent series of experiments with different batches of cells.
(A) Analysis of the amount of radioligand 125I-Tyr-flg22 remaining free in cell suspension. Supernatant was freed of cells by filtration after 1, 5, and 20 min of incubation as indicated and analyzed by reversed-phase HPLC on a C18 column. Radioactivity in the eluate was detected with an online radioactivity detector (Raytest Ramona-90; Raytest Isotopenmessgeräte, Straubenhardt, Germany). Shaded area indicates the time of elution for untreated radioligand 125I-Tyr-flg22.
(B) Analysis of radioligand bound to cells. After incubation with radioligand for 5 or 120 min as indicated, cells were washed as described for the binding assays. Radioactivity bound to cells was released by treatment under acidic conditions (0.5 min at pH 2.0) and analyzed by HPLC as described in (A).