Activity of E5564 after different conditions of preincubation and dissociation incubation with cells
| Time of Dissociation | Activated (IC50) of E5564 (in nM) after Incubation with RAW 264 Cells Preincubated in Absence (Pre−) or Presence (Pre+) of Serum Followed by disseciation Incubation in Absence (Post−) or Presence (Post+) of Serum | |||
|---|---|---|---|---|
| Pre−/Post− | Pre+/Post− | Pre−/Post+ | Pre+/Post+ | |
| h | ||||
| 0 | 0.8 ± 0.1 | 1.1 ± 0 | 0.8 ± 0.1 | 1.1 ± 0 |
| 0.5 | 0.7 ± 0.1 | 2.5 | 1.1 | 5.1 |
| 2 | ∗ | ∗ | 25.0 | 24.5 |
Raw 264.7 cells were incubated for 1 h in the presence or absence of 10% fetal bovine serum and in the absence or presence of E5564 at 100, 33, 11, 3.3, and 1.1 nM. Cells were then washed three times in serum-free medium, incubated in the same media with or without added fetal calf serum (10%) for the indicated period of time, washed again, and assayed for LPS-mediated generation of TNF-α as described underMaterials and Methods.
4-178 Poorly reproducible. Incubation in serum-free media for an extended period of time combined with extensive washing resulted in loss of adherent cells and cell death. Cytokine release was low and variable.