normally These data illustrate the complexity of the IL 1B mediated astrocyte Inhibitors,Modulators,Libraries inflammatory response and provide details of the regulatory mechanisms involved. Experimental Inhibitors,Modulators,Libraries procedures Isolation, cultivation and activation of human astrocytes Human astrocytes Inhibitors,Modulators,Libraries were isolated from first and early second trimester aborted spe cimens obtained from the Birth Defects Laboratory, University of Washington, Seattle, in full compliance with the ethical guidelines of the NIH, the Universities of Washington and North Texas Health Science Center. The Birth Defects Laboratory is an NIH funded institu tion with the mission of disseminating tissues for the ad vancement Inhibitors,Modulators,Libraries of biomedical research. Astrocytes were isolated from specimens as originally described earlier. Activation of astrocytes was achieved by applying IL 1B for various time intervals.
IL 1B is a prototypical inflammatory cytokine expressed during HIV 1 CNS in fection, making IL 1B an excellent choice for studying human astrocyte function during neuroinflammation. We empirically tested IL 1B dosage and transfec tion of astrocytes with C EBPB small interfering RNA as described in Fields et al. Accordingly, these data provide relevant implications Inhibitors,Modulators,Libraries for astrocyte function in many pathologies involving neuroinflammation. RNA isolation and TaqManW human inflammation array and real time reverse transcription polymerase chain reaction Astrocytes were transfected with nonspecific control or C EBPB specific siRNA by nucleo fection and then cultured as adherent monolayers in 75 cm2 flasks at a density of 8 �� 106 cells per flask or six well plates at a density of 1.
6 �� 106 cells per well for 24 h. The following day, the medium was exchanged with or without IL 1B for 24 h. Alternatively, astrocytes were cultured in six well plates at a density of 1. 6 �� 106 cells per well for 24 h, and then the medium was exchanged with or without MAPK selective inhibitors for 1 h and then with inhibitor plus IL 1B for 12 h. Astrocyte RNA was extracted www.selleckchem.com/products/BAY-73-4506.html and reverse transcribed into cDNA as per the manufacturers instruc tions. The TaqManW Human Inflammation Array was used to assay for expression of 92 inflammation genes using total RNA from two independent human astrocyte donors. Data are illustrated in Figure 1 as a heat map, red and green represent positive and negative fold change, respectively. Increasing color intensity corresponds to in creasing absolute value. Orange and blue represent positive and negative percent change, respectively, in target MRNA levels in siC EBPB IL 1B versus IL 1B conditions. Read outs from each TaqManW assay target were independently analyzed comparing control, IL 1B and siC EBPB IL 1B by one way analysis of variance followed by Newman Keuls post test.