Allergy 41, 1346C1359. and largely coexpress in the same subpopulation of PMN. The mRNA expression and the size of subpopulations expressing certain chemosensory receptors varied largely among individual blood samples, suggesting a regulated expression of olfactory and taste receptors in these cells. Moreover, we show mRNA expression of their downstream signaling molecules and demonstrate that PTX abolishes saccharin- or 2-PEA-induced PMN chemotactic migration, indicating a role for Gi-type proteins. In summary, our data suggest “chemosensory”-type subpopulations of circulating leukocytes. = 47; Bavarian Red Cross Blood Bank, Munich, Germany). Bovine PMN were isolated from peripheral blood, obtained from 6 healthy female Brown Suisse cows (Research Station Veitshof, Physiology Weihenstephan, Freising, Germany). Human and bovine blood leukocytes were purified by use of Ficoll density gradient centrifugation, as described previously [22]. Blood leukocyte-positive selection was performed by use of anti-CD14+ (monocytes), -CD3+ (T cells), -CD19+ (B cells), and -CD56+ (NK cells) magnetobead-conjugated antibodies from Miltenyi Biotec [22]. The purity of isolated leukocytes was confirmed by flow cytometry (MACSQuant analyzer; Miltenyi Biotec) and was always 90%. The cells were resuspended further at 1 106/ml concentration in RPMI 1640 or in GITC buffer [22] for RNA isolation. RNA isolation and cDNA synthesis Total RNA from blood leukocytes was isolated and purified by CsCl gradient centrifugation, as described previously [22]. Moreover, to avoid genomic DNA contamination, the RNA preparation was followed by a DNAse-I (Baseline-Zero; Biozym, Hessisch Oldendorf, Germany) digestion. The quality of Amisulpride hydrochloride RNA was tested in 12 randomly selected humans and in all 6 bovine RNA samples by Experion automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) by use of standard sense RNA analysis LabChips, according to the manufacturers instructions (Supplemental Fig. 1). The quality of tested RNA samples was determined by the RNA quality index, which ranged between 7.8 and 9.7. cDNA was synthesized from total RNA by use of the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturers recommendations. RT-PCR The presence of RNA transcripts for human Class I and Course II OR was looked into primarily Amisulpride hydrochloride by RT-PCR in every 5 cell types by Amisulpride hydrochloride usage of models of 8- to 512-collapse degenerate oligonucleotide primers (Biomers; Supplemental Components and Strategies and Supplemental Desk 1), built to detect extremely conserved areas within Course I or Course II OR at the start of TMII or the finish of TMIII. PCR reactions had been performed by using GoTaq PCR Get better at Mix (1/2 response quantity; Promega, Mannheim, Germany), based on the producers recommendations. The existence and specific distribution of 55 Course I OR, 27 Course II OR, 25 TAS2R, and 3 TAS1R in various bloodstream cells had been examined by RT-PCR by usage Amisulpride hydrochloride of cDNA further, as Rabbit Polyclonal to TISD referred to previously (ref. [22] and in Supplemental Components and Strategies). Sequences for many gene-specific primers (Eurofins MWG) are detailed in Supplemental Components and Methods. The power of primers to allow receptor-specific amplicons was examined by PCR by usage of genomic or plasmid DNA (Supplemental Figs. 2C4). The identification of human being and bovine Course I OR, TAS2R, and TAS1R was verified by Sanger sequencing of 50% (arbitrarily selected) of most agarose gel-purified amplicons. RT-PCR data had been examined as percentage of positive Course I OR-, TAS2R-, or TAS1R-specific amplicons/cell type and across all bloodstream samples, as seen in agarose gel electrophoresis. RT-qPCR RNA manifestation level of human being and bovine orthologs for Course I OR, TAS2R, and TAS1R in PMN was quantitatively weighed against RT-qPCR (= 15, and = 6, respectively) as comparative manifestation, normalized to typically 6 chosen and stably indicated guide genes (GAPDH, RNA polymerase II A, 0.05). PMN migration within RPMI-1640 moderate was utilized as a poor control. PMN transmigration assays had been.