dna sequences encoding full length oa ox40 and oa cd27  (GenScript corporation)

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: Colocalisation of the ovine OX40L and CD70 ligands with their cognate receptors. ( A ) Immunolocalisation of Oa OX40 and Oa CD27 in transfected HEK293 cells detected by anti-V5 tag antibody and counterstained with DAPI. Cells expressing ( B ) Oa OX40 or ( D ) Oa CD27, were incubated with 10 μg of ( B ) purified r Oa OX40L or ( D ) purified r Oa CD70 for 15 min before fixation and immunofluorescence. Oa OX40 and Oa CD27 expression was detected using anti-V5 tag antibodies (red); r Oa OX40L and r Oa CD70 presence were detected with an anti-ovine Fc antibody (green). ( B , D ) Insets show detail of colocalisation in each case. Merge and orthogonal projection were obtained with ImageJ software. Scale bar = 10 μm, indent scale bar = 4 μm. In panel ( C ) and ( E ), an enlarged image of corresponding insets in Merge and fluorescence intensity profiles for the indicated white lines on the images are shown. ( F ) ImageJ Image calculator function was used to evaluate colocalisation of ligand signal with its receptor. Representative images of colocalisation analysis in a Z-plane (presented as 16LUT signal) are shown. The ImageJ calculator tool was used for pixel colocalisation for the fluorescence channels of ligands (r Oa OX40L or r Oa CD70) and their respective receptors (r Oa OX40 or r Oa CD27). ( G ) The percentage of recombinant ligand signals colocalising with their receptors in ( F ) was analysed in 25–40 cells for each condition. Mean ± SD are indicated for each condition.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Expressing, Incubation, Purification, Immunofluorescence, Software, Fluorescence, Recombinant

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: The secreted r Oa OX40L and r Oa CD70 expressed from recombinant adenovirus-infected cells colocalise with their cognate ovine receptors. Cells expressing ( A ) Oa OX40 or (C) Oa CD27, were incubated with conditioned media from cells infected with ( A , B ) Ad5- Oa OX40L (r Oa OX40L) or ( C , D ) Ad5- Oa CD70 (r Oa CD70) for 15 min before fixation and immunofluorescence. r Oa OX40L and r Oa CD70 were detected with an anti-Fc antibody (green); Oa OX40 and Oa CD27 expression was detected using anti-V5 tag antibodies (red). ( B , D ) Insets show detail of colocalisation for each case. Merge and orthogonal projections were obtained with ImageJ software. Scale bar = 10 μm, indent scale bar = 4 μm. In panel ( B ) and ( D ), an enlarged image of corresponding insets in Merge and fluorescence intensity profiles for the indicated white lines on the images are shown. ( E ) Representative images of colocalisation analysis in a Z-plane (presented as 16LUT signal) are shown. The ImageJ calculator tool was used for pixel colocalisation for the fluorescence channels of ligands (r Oa OX40L or r Oa CD70) and their respective receptors (r Oa OX40 or r Oa CD27). ( F ) The percentage of ( E ) adenovirus-produced ligands signal colocalising with their receptors was analysed in 25–40 cells for each condition. Mean ± SD are indicated for each condition.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Recombinant, Infection, Expressing, Incubation, Immunofluorescence, Software, Fluorescence, Produced

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: The r Oa OX40L and r Oa CD70 specifically elicit signalling through their cognate receptors Oa OX40 and Oa CD27. HEK293-pr(IFNB)-GFP cells were transfected with, ( A – C ) Oa OX40-V5 and ( D – F ) Oa CD27-V5. At 24 h post transfection, the cells were stimulated with 10 μg of purified ( A ) rOa OX40L or ( D ) rOa CD70 proteins, or equivalent amounts of conditioned media from Ad5- Oa OX40L ( B ) rOa OX40L, ( E ) Ad5- Oa CD70, or ( C , F ) Ad5-DsRed infected Vero cells proteins in conditioned media from Ad5- Oa OX40L, Ad5- Oa CD70, or Ad5-DsRed infections. At 16 h post stimulation, the cells were fixed and an immunofluorescence using anti-V5 (red) to detect the transfected receptors was performed. GFP (green) activity was detected by fluorescence microscopy and DAPI was used to counterstain cells to nuclei (blue). Bars = 20 µm.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Purification, Infection, Immunofluorescence, Activity Assay, Fluorescence, Microscopy

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: Dose dependent induction of signalling by r Oa OX40L and r Oa CD70 ligands. HEK-293/prIFNB-GFP cells were transfected with plasmids to express the receptors Oa OX40 or Oa CD27 and stimulated at 24 h post transfection with three equivalent and increasing doses (1×, 2×, and 4×) of conditioned media from Ad5- Oa OX40L (r Oa OX40L) or Ad5- Oa CD70 (r Oa CD70) infected Vero cells. As a positive control of signalling induction, untransfected cells were infected with Sendai virus (SeV) at a multiplicity of infection (moi) of 1 pfu/cell for 16 h. GFP expressing cells were counted on an immunofluorescence microscope on 12 randomly chosen fields and % of GFP expressing cells for each field are plotted. Mean ± SD for each condition are indicated with bars.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Transfection, Infection, Positive Control, Virus, Expressing, Immunofluorescence, Microscopy

Journal: Vaccines

Article Title: Activation of OX40 and CD27 Costimulatory Signalling in Sheep through Recombinant Ovine Ligands

doi: 10.3390/vaccines8020333

Figure Lengend Snippet: CD27 expression on ovine PBMC populations. Ovine PBMC, derived from n = 15 animals were costained for CD27 and CD4, CD8, CD335, or B cell markers. Gates were set using the corresponding isotype controls. Isotype control staining and representative dot plots for CD27 staining and CD4; CD8; CD335 or B cell marker are shown. Bar charts show the mean (±SD) percentage of CD27 + and CD27 - cells in the CD4/CD8/CD335/B cell marker gates (upper quadrants) in donor sheep PBMC.

Article Snippet: The DNA sequences encoding full length Oa OX40 and Oa CD27 were optimised for expression in mammalian cells and synthesised in vitro (GenScript, Piscataway, NJ, USA).

Techniques: Expressing, Derivative Assay, Control, Staining, Marker