The tissue distribution of the human b b 3 -adrenoceptor studied using a monoclonal antibody: Direct evidence of the b b 3 -adrenoceptor in human adipose tissue, atrium and skeletal muscle 2,3 The human b 3 -adrenoceptor was later cloned by Emorine et al. 4 The pharmacology of the cloned b 3 -adrenoceptor agreed with the pharmacological data previously obtained in rodent adipose tissue, gut, and skeletal muscle in that the badrenoceptors in these tissues were insensitive to classical b-adrenoceptor antagonists such as propranolol.5 ± 8 Aryloxypropanolamine b 1 ab 2 adrenoceptor antagonists, exempli®ed by CGP12177, evoke a lipolytic response in rat adipose tissue through agonism at b 3 -adrenoceptors. 9 Similarly, selective b 3 -adrenoceptor agonists, such as BRL-37344, showed similar or greater potency than isoproterenol in stimulating lipolysis, but were much less potent than isoproterenol in stimulating responses mediated by b 1 -or b 2 -adrenoceptors. 10 The assessment of the pharmacological role of b 3 -adrenoceptors in human tissues has proved more controversial. For example, lipolysis in human white adipocytes induced by isoproterenol is sensitive to propranolol.11 Also, CGP12177-induced lipolytic responses have been demonstrated by some 12 ± 14 but not others. 9,15 It is now evident from comparisons of cloned human and rat b 3 -adrenoceptors that there are signi®cant species differences in pharmacology. 16 Attempts to detect b 3 -mRNA in human tissues using reverse-transcription PCR have also given conicting results. Krief et al 17 detected b 3 -adrenoceptor mRNA in several tissues, including gall bladder, adipose tissue and colon, while Thomas and Liggett 18 failed to detect a b 3 -adrenoceptor signal. Recently, RNAase protection assays, that do not rely on ampli®cation techniques, were used to identify b 3 -adrenoceptor mRNA in a variety of human tissues, including

The discrimination of apoptotic and normal rat thymocytes by forward-angle light scatter in the W depended on the angle over which the scattered light was collected. To achieve good separation, the light needed to be collected over a narrow angle. It was also observed that apoptotic thymocytes gave less forward light scatter than normal cells, whereas, under identical conditions, apoptotic cells from a murine cell line gave more light scatter than their normal counterparts. We suggest that the forward light scatter of apoptotic cells is affected by several factors, including changes in cell size and in the internal structure of the cell.o 1995 wiley-Liss, hc. Key terms: Flow cytometry, light scatter, apoptosisOne of the features of apoptotic cell death is cell shrinkage (8,13,31). Several investigators have reported that unfixed apoptotic cells scattered less light than normal cells, both at 488 nm (4,10,11,24) and at 360 or 350 nm (3,5,16,17,30). We found that, by using an Ortho Cytofluorograf with krypton laser-emitting UV light (350 nm), apoptotic rat thymocytes could be separated from normal cells by forward-angle light scatter (FALS) alone (3,17,30). By contrast, apoptotic cells from a murine haemopoietic cell line, under the same conditions, gave more FALS than normal cells (16).When we were evaluating the performance of two other flow cytometers (Becton-Dickinson Vantage and Coulter Elite), we found there was little discrimination between apoptotic and normal thymocytes when using the normal settings on FALS. However, when the measurement of FALS was restricted to the smallest possible angle, a good separation was obtained on the Coulter Elite and on the Becton-Dickinson FACStar.We report these results to assist other workers separating cells of different sizes on the basis of light scatter and as an illustration of the different factors involved in light scattering. MATERIALS AND METHODS CellsImmature rat thymocytes were prepared as described elsewhere (19). After incubation for 4 h at 37°C with either 10 pM etoposide or 0.1 p M dexamethasone, the cells were labelled with Hoechst 33342 (1 pg/ml at 37°C for 10 min) and propidium iodide (PI; 5 pg/ml) (3,16,17,30) and kept on ice until they were analysed.Apoptosis was induced in a cell line derived from murine haemopoietic cells (BGF3; 18) by incubating cells in growth medium without the growth factor, interleukin-3 (IL-3), for 16 h (20). Cells were labelled by incubation with Hoechst 33342 (1 pg/ml at 37°C for 7 min) and PI Flow CytometryData were recorded on an Ortho Cytofluorograf 50H with a krypton laser tuned to give 100 mW at 350 nm, on a Coulter Elite with a helium-cadmium laser (25 mW at 325 nrn), or on a Becton-Dickinson FACStar with a Coherent argon-ion laser (100 mW at 360 nm). All three instruments were equipped with quartz cuvettes as flow cells. With all three instruments, FALS and blue (460 nm) and red (>630 nm) fluorescences were recorded. After gating out the cells fluorescing red (PI positive), a bivariate histogram (cytogram) of blue flu...

In order to promote the killing of tumour cells by antibody a derivative has been synthesized in which Fab'gamma from xenogeneic antibody is thioether-bonded to half-cystine on normal IgG of the species to be treated. The resulting entity, FabIgG, is obtained with about a 40% yield of the starting Fab'gamma. Being univalent it evades antigenic modulation. It activates complement efficiently, is minimally immunogenic, and appears to be catabolized at the slow rate characteristic of autologous IgG.

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