A pulse of TNF promotes angiogenesis in vivo. Mice (15) were injected intradermally with Matrigel (500 μL) and then split randomly into 3 groups. One group received daily injections into the gel of PBS for 7 days, one group received TNF (1 ng) for 3 days, followed by PBS for 4 days, and the third received daily injections of TNF (1 ng) for 7 days. Skin containing the gel was then harvested and examined by immunohistochemistry for the presence of blood vessels (CD31: pink) and nuclei (DAPI: blue). Images were captured on an Olympus IX70 inverted phase contrast/fluorescence microscope using a 40×/0.60 NA fluorescence objective and an Optronics digital camera. High-resolution images were magnified in Photoshop. (A) Representative sections from each of the groups. (B) The number of ECs present was determined by counting pink (CD31⁺) cells and this was normalized to area of tissue as determined by DAPI staining. Shown are means and SD of CD31⁺ cells per unit area. *P < .005 relative to vehicle control; **P < .05 relative to vehicle control, by Student t test. (C) EC-coated beads were established in fibrin gels and incubated with conditioned medium (CM) from either resting or activated PBMCs (CD3 + CD28 for 48 hours). Cultures either received a pulse of CM (2 days of “activated” CM followed by 4 days of “resting” CM) or continuous CM (resting or activated). All cultures had medium changed every 2 days. At 8 days sprouts were counted. Means and SD are shown. *P < .001 for resting compared with activated/continuous; **P < .001 for resting compared with activated/pulse, by Student t test. One of 3 similar experiments.