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human foreskin fibroblasts  (ATCC)
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ATCC human foreskin fibroblasts
Fig. 1. Downregulation of UL54 expression by targeted siRNAs. (A) Experimental set-up. Subconfluent cultures of human primary foreskin fibroblasts were withdrawn from the cell cycle by growth factor deprivation. After 48 h, cells were trans- fected with different siRNAs. At 24 h p.i., cells were infected with HCMV (m.o.i.=10). Cells and supernatants were harvested separately at the indicated time points and processed for further analysis. (B) Total RNA from 24 h p.i. was prepared and analysed for UL54 mRNA expression by ribonuclease protection analysis. An L32-specific probe was included in the same assay as a loading control. Relative expression levels were quantified and given as percentages. (C) Expression levels of the immediate-early protein 1 (IE1) were determined by immuno- blot analysis of whole-cell lysates. A non-specific background band occurring in all lanes is marked with an asterisk and indicates equal gel loading.
Human Foreskin Fibroblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Fig. 1. Downregulation of UL54 expression by targeted siRNAs. (A) Experimental set-up. Subconfluent cultures of human primary foreskin fibroblasts were withdrawn from the cell cycle by growth factor deprivation. After 48 h, cells were trans- fected with different siRNAs. At 24 h p.i., cells were infected with HCMV (m.o.i.=10). Cells and supernatants were harvested separately at the indicated time points and processed for further analysis. (B) Total RNA from 24 h p.i. was prepared and analysed for UL54 mRNA expression by ribonuclease protection analysis. An L32-specific probe was included in the same assay as a loading control. Relative expression levels were quantified and given as percentages. (C) Expression levels of the immediate-early protein 1 (IE1) were determined by immuno- blot analysis of whole-cell lysates. A non-specific background band occurring in all lanes is marked with an asterisk and indicates equal gel loading.

Journal: The Journal of general virology

Article Title: Inhibition of human cytomegalovirus replication by small interfering RNAs.

doi: 10.1099/vir.0.19453-0

Figure Lengend Snippet: Fig. 1. Downregulation of UL54 expression by targeted siRNAs. (A) Experimental set-up. Subconfluent cultures of human primary foreskin fibroblasts were withdrawn from the cell cycle by growth factor deprivation. After 48 h, cells were trans- fected with different siRNAs. At 24 h p.i., cells were infected with HCMV (m.o.i.=10). Cells and supernatants were harvested separately at the indicated time points and processed for further analysis. (B) Total RNA from 24 h p.i. was prepared and analysed for UL54 mRNA expression by ribonuclease protection analysis. An L32-specific probe was included in the same assay as a loading control. Relative expression levels were quantified and given as percentages. (C) Expression levels of the immediate-early protein 1 (IE1) were determined by immuno- blot analysis of whole-cell lysates. A non-specific background band occurring in all lanes is marked with an asterisk and indicates equal gel loading.

Article Snippet: We therefore set out to optimize the standard transfection protocol from the Tuschl laboratory (Elbashir et al., 2002) for use in serum-starved primary human foreskin fibroblasts (CRL-2429 from ATCC) employing the transfection reagent Oligofectamine (Invitrogen).

Techniques: Expressing, Infection, Control

Fig. 2. Inhibition of virus replication by UL54-directed RNA interference. Quiescent fibroblasts were control transfected (+siEGFP), transfected with siUL54(1), or left untransfected. Subsequently, cell cultures were infected with HCMV (m.o.i.=10). Where indicated, 50 mM ganciclovir was added after virus adsorption. (A) At the indicated time points, cells were stained with propidium iodide and analysed for DNA content by flow cytometry. DNA histograms contained no cell doublets and were brought to equal cell numbers. (B) An overlay of the different DNA profiles observed at 72 h p.i. (see main text for further explanation). (C) Supernatants of the infected cultures were collected and virus titres were determined twice in duplicate plaque assays.

Journal: The Journal of general virology

Article Title: Inhibition of human cytomegalovirus replication by small interfering RNAs.

doi: 10.1099/vir.0.19453-0

Figure Lengend Snippet: Fig. 2. Inhibition of virus replication by UL54-directed RNA interference. Quiescent fibroblasts were control transfected (+siEGFP), transfected with siUL54(1), or left untransfected. Subsequently, cell cultures were infected with HCMV (m.o.i.=10). Where indicated, 50 mM ganciclovir was added after virus adsorption. (A) At the indicated time points, cells were stained with propidium iodide and analysed for DNA content by flow cytometry. DNA histograms contained no cell doublets and were brought to equal cell numbers. (B) An overlay of the different DNA profiles observed at 72 h p.i. (see main text for further explanation). (C) Supernatants of the infected cultures were collected and virus titres were determined twice in duplicate plaque assays.

Article Snippet: We therefore set out to optimize the standard transfection protocol from the Tuschl laboratory (Elbashir et al., 2002) for use in serum-starved primary human foreskin fibroblasts (CRL-2429 from ATCC) employing the transfection reagent Oligofectamine (Invitrogen).

Techniques: Inhibition, Virus, Control, Transfection, Infection, Adsorption, Staining, Cytometry