In situ hybridization for cytokine mRNA with digoxigenin-labeled riboprobes Sensitivity of detection and double label applications

Karr, Laurel J.; Panoskaltsis‐Mortari, Angela; Li, Jimin; Devore-Carter, Denise; Weaver, Casey T.; Bucy, R. Pat

doi:10.1016/0022-1759(95)00027-8

Cited by 26 publications

(16 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Transcription was performed in the presence of 0n04 M Tris-HCl pH 8n0, 6 mM MgCl # , 10 mM dithiothreitol, 2 mM spermidine, 10 mM NaCl, 0n1 unit RNase inhibitor, 1 mM each of ATP, GTP and CTP, 0n65 mM UTP and 0n35 mM digoxigenin-11-UTP (DIG-11-UTP). Following transcription, the mixture was treated with DNase, phenol-extracted, ethanol-precipitated, and the RNA suspended in RNase-free water and subjected to alkaline hydrolysis in carbonate buffer (pH 10n2) for 20 min at 60 mC (Karr et al, 1995). After neutralization with acetic acid, the fragments of 100-150 nucleotides were precipitated in ethanol and resuspended in RNase-free water.…”

Section: Methodsmentioning

confidence: 99%

Cell-to-cell spread of poliovirus in the spinal cord of bonnet monkeys (Macaca radiata).

Ponnuraj

John

Levin

et al. 1998

Journal of General Virology

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Cell-to-cell spread of poliovirus in the spinal cord of bonnet monkeys (Macaca radiata).

Ponnuraj

John

Levin

et al. 1998

Journal of General Virology

View full text Add to dashboard Cite

“…Taken together, these data suggest that digoxigenin-labelled riboprobes may be more sensitive than 35 S-labelled riboprobes. Certainly, a recent report showed that digoxigenin-labelled riboprobes are at least as sensitive as 35 S-labelled riboprobes when used to detect interleukins in T-cells [20].…”

Section: Cellular Localization Of Tgf-β 1 and Tgf-β 3 Mrna Transcriptmentioning

confidence: 99%

Diverse cellular TGF-beta 1 and TGF-beta 3 gene expression in normal human and murine lung

Coker¹,

Guy²,

Shahzeidi³

et al. 1996

Eur Respir J

View full text Add to dashboard Cite

D Di iv ve er rs se e c ce el ll lu ul la ar r T TG GF F--β 1 1 a an nd d T TG GF F--β 3 3 g ge en ne e e ex xp pr re es ss si io on n i in n n no or rm ma al l h hu um ma an n a an nd d m mu ur ri in ne e l lu un ng g This procedure was technically simple, providing excellent resolution. TGF-β 1 and TGF-β 3 messenger ribonucleic acid (mRNA) transcripts were detected in a wide variety of cells. In human lung, mRNA for both isoforms was localized to bronchiolar epithelium and alveolar macrophages. TGF-β 1 , but not TGF-β 3 mRNA was detected in mesenchymal and endothelial cells. In murine tissue, TGF-β 1 , mRNA was localized to bronchiolar epithelium, Clara cells, mesenchymal cells, pulmonary endothelium and alveolar cells, including macrophages. TGF-β 3 mRNA was similarly distributed but not detected in endothelium.In summary, using a nonisotopic technique in lung tissue, we have detailed the cells expressing the transforming growth factor-β 1 and β 3 genes in human and murine lung. There was widespread expression of these cytokines in normal lung consistent with autocrine or paracrine roles in regulating cellular turnover, immune defence and matrix protein metabolism.

show abstract

“…The ISH technique is described in detail elsewhere (17,18 Table 1). The time of peak expression for cytokine mRNAs was determined ( Fig.…”

Section: Naive Af3 T-cell Receptor (Tcr) Transgenic T Cells Activatedmentioning

confidence: 99%

“…The ISH technique is described in detail elsewhere (17,18). The probes were antisense, single-stranded RNA molecules with the following sequences: IL-2, 237-837; IL-4, 1-393; IL-5, 1-1534; IL-10, 392-937; IFN-,y, 1-270.…”

Section: Introductionmentioning

confidence: 99%

Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.

Bucy

Karr

Huang

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

131

View full text Add to dashboard Cite

CD4+ T cells from a8-T-cell receptor transgenic mice were analyzed for coexpression of cytokine mRNAs during phenotype development using a double-label in situ hybridization technique. T cells that produced cytokines in the primary response were a fraction of the activated population, and only a minority of the cytokine-positive cells coexpressed two cytokines. In secondary responses, frequencies of doublepositive cells increased, although they remained a minority of the total. Of the cytokine pairs examined, interleukin (IL)-4 and IL-5 were the most frequently coexpressed. IL-4 and interferon y showed the greatest tendency toward segregation of expression, being rarely coexpressed after the primary stimulation. These data indicate that there is significant heterogeneity of cytokine gene expression by individual CD4+ T cells during early antigenic responses. Coexpression of any pairs of cytokines, much less Thl and Th2 cytokines, is generally the exception. The ThO phenotype is a population phenotype rather than an individual cell phenotype.Regulation of immune responses by CD4+ T cells is mediated by antigen-induced production of cytokines. Naive CD4+ T cells have limited cytokine responses; hence, their regulatory capacity and effector function are limited (1-3). After antigendriven differentiation, there is significant diversification of CD4+ T-cell cytokine responses (3). At least two differentiation pathways can be distinguished-the so-called Thl/Th2 paradigm (4-6). Recent studies have begun to elucidate the mechanisms that control CD4+ T-cell phenotype development (3, 7-10) and have established a principal role for cytokines as determinants of Thl vs. Th2 development. Interleukin (IL)-4, IL-12, IL-10, interferon (IFN)-,y, and transforming growth factor ,B exert critical effects on phenotype development, albeit by distinct mechanisms (7-9, 11, 12 (14), who produced transgenic mice with a thymidine kinase (TK) cDNA linked to the IL-4 promoter and showed that in the presence of ganciclovir, the mitogen-stimulated development of both IL-4-and IFN-y-producing T-cell effectors was blocked. These data suggest that an IL-4-producing intermediate precedes development of both Thl and Th2 cells.In an effort to dissect single cell responses during development of cytokine phenotypes in an a,B-TCR transgenic system, we have developed a double-label in situ hybridization (ISH) method to detect the expression of pairs of cytokine mRNAs in individual cells. Our results demonstrate a marked heterogeneity of cytokine gene expression during primary and subsequent antigenic stimulations and indicate that coexpression of cytokines by the same cell is the exception. These data suggest that the ThO-like phenotype in early CD4+ T-cell differentiation is a population phenotype rather than an individual cell phenotype and emphasize the independent regulation of each of the cytokine genes.EXPERIMENTAL PROCEDURES Animals. The DO10 transgenic line has been described (15). These animals express a transgenic TCR with specificity...

show abstract

In situ hybridization for cytokine mRNA with digoxigenin-labeled riboprobes Sensitivity of detection and double label applications

Cited by 26 publications

References 20 publications

Cell-to-cell spread of poliovirus in the spinal cord of bonnet monkeys (Macaca radiata).

Cell-to-cell spread of poliovirus in the spinal cord of bonnet monkeys (Macaca radiata).

Diverse cellular TGF-beta 1 and TGF-beta 3 gene expression in normal human and murine lung

Single cell analysis of cytokine gene coexpression during CD4+ T-cell phenotype development.

Contact Info

Product

Resources

About