title: Detection of in vivo-induced IL-1 mRNA in murine cells by flow cytometry (FC) and fluorescent in situ hybridization (FISH).
abstract: Flow cytometry (FC) and fluorescent in situ hybridization (FISH) were used to detect in vivo induced IL-1 alpha (a) messenger RNA. Spleen cells and thioglycollate-induced peritoneal exudate cells (PEC) were harvested from Balb/C mice three hours after intraperitoneal injection of 20 micrograms LPS. Cells from each population were phenotyped for MAC or IAd, fixed in 4% paraformaldehyde and then permeabilized with 70% ETOH. RNA-RNA FISH was performed by incubating suspended cells at 37 degrees C for 17 hours with biotinylated sense IL-1a, antisense IL-1a or antisense IL-2 probes in 50% formamide. Hybridized cells were washed in 2X SSC, treated with RNAse, stained with avidin conjugated to fluorescein (FITC) or allophycocyanin (APC) and analyzed immediately by FC. Initially, avidin-FITC was used to detect hybridized probe. Dual fluorescent FC analysis of IL-1a mRNA expression in LPS stimulated IAd+ cells showed an increase in mean fluorescent intensity (MFI) of 51 log channels (0.25 logs) when compared to unstimulated cells. Additionally, induction of specific IL-1a mRNA expression in cells from LPS treated animals was illustrated by increases in percent positive cells (24%) and in equivalent soluble fluorescein molecules (ESFM) bound (47%) when compared to cells from vehicle treated mice. Unhybridized cells and cells hybridized with control antisense IL-2 probe did not exhibit increases in MFI or ESFM. In subsequent experiments on MAC+ PEC, the use of avidin-APC to detect bound probe resulted in a greater separation between the FISH signals of antisense IL-1a and control sense probe (121 log channels, 0.6 logs) than that seen with FITC.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Schering-Plough Research, Bloomfield, N.J. 07003.
authors.forename: K J
authors.initials: KJ
authors.lastname: Pennline
authors.forename: F
authors.initials: F
authors.lastname: Pellerito-Bessette
authors.forename: S P
authors.initials: SP
authors.lastname: Umland
authors.forename: M I
authors.initials: MI
authors.lastname: Siegel
authors.forename: S R
authors.initials: SR
authors.lastname: Smith
mesh_terms: D000818:Animals; D016375:Antisense Elements (Genetics); D001202:Ascitic Fluid / Q000378:metabolism* / Q000473:pathology; D001360:Avidin / Q000737:chemistry; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics*; D008070:Lipopolysaccharides / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009693:Nucleic Acid Hybridization; D010798:Phycocyanin / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D013154:Spleen / Q000166:cytology / Q000378:metabolism*
chemical_list: D016375:Antisense Elements (Genetics); D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; C002497:allophycocyanin; D010798:Phycocyanin; D001360:Avidin; D016650:Fluorescein-5-isothiocyanate