Cell death (A), DNA fragmentation (B), and FasL mRNA expression (C) are increased on CD28 crosslinking. (A) DWT6.11 cells were seeded for 12 hours in a 24-well plate in the presence of the indicated MoAbs and PI stained followed by cytofluorometry as described in the Materials and Methods. The results are presented as averaged percentage of subdiploid nuclei obtained from three independent experiments (±SD). (B) DNA was extracted from the cells collected at 8 hours of incubation and analyzed on a 1% agarose gel containing ethidium bromide.(C) The cells were collected after a 6-hour incubation period, followed by mRNA extraction, reverse transcription, and PCR using FasL, IL-2, or β2 microglobulin primer pairs as described in the Materials and Methods. Amplification products were analyzed on a 1% agarose gel followed by ethidium bromide staining. Integrated intensity of the specific band was determined using the BioImage (Millipore Corp). Results were normalized to the relative levels of β2m and are presented as histograms. One representative experiment out of two is shown. Where indicated, the X symbol indicates that goat antimouse polyclonal antiserum was added.