primer express software  (Thermo Fisher)


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    Primer Express Software v3 0 1 License
    Description:
    The Primer Express Software v3 0 1 allows you to design your own primers and probes using TaqMan and SYBR Green I dye chemistries for gene quantitation and allelic discrimination SNP real time PCR applications Developed specifically for use with our StepOne StepOnePlus 7300 7500 7500 Fast 7900HT ViiA 7 and QuantStudio real time PCR systems Primer Express Software provides customized application specific documents for absolute⁄relative quantitation and allelic discrimination • Enables individual oligonucleotide design for real time PCR applications • Supports assays based on TaqMan and SYBR Green I dye chemistries • Provides design flexibility ease of use and requires minimal optimization • Provides robust assay performance when used in accordance with Rapid Assay Development Guidelines RADG Easy to Learn SoftwareThe Primer Express Software v3 0 1 is flexible easy to learn and to use and requires minimal optimization It includes an algorithm optimized specifically for use with TaqMan based reagents recommended primer⁄probe concentrations and default thermal cycling conditions Primer Express Software also supports assays based on the SYBR Green I dye This minimizes the need for assay optimization and streamlines the design process The software also allows customized designs if the probe and⁄or one or more primers are known Default and adjustable parameters Figure 1 provide the flexibility required for all users regardSimple to UseSimplicity and flexibility are the hallmarks of Primer Express Software v3 0 1 which is fully compatible with all StepOne StepOnePlus 7300 7500 7500 Fast 7900HT ViiA 7 and QuantStudio real time PCR systems Whether your research includes gene quantitation or allelic discrimination studies Primer Express Software belongs in your lab Automated and Manual Primer⁄Probe DesignThe Primer Express Software v3 0 1 will design your primers and probes automatically or you can design them manually For automated design simply choose either TaqMan or TaqMan MGB assays If you know the sequence you need only to import or copy and paste the sequence and quickly view your results Alternatively if you prefer to design the primers and probes manually the procedure is equally simple and straight forward as well Rapid Assay Development GuidelinesThe Primer Express Software v3 0 1 was designed with our Rapid Assay Development Guidelines RADG The RADG are a simple yet comprehensive set of guidelines for use with our real time PCR instruments and reagents Many traditional variables including magnesium chloride concentration and the thermal cycling protocol itself have been standardized which substantially reduces assay development time For Research Use Only Not for use in diagnostics procedures
    Catalog Number:
    4363991
    Price:
    None
    Category:
    Software and Digital Storage
    Applications:
    PCR & Real-Time PCR|Real Time PCR (qPCR)|Real-Time PCR Instruments, Software & Calibration
    Buy from Supplier


    Structured Review

    Thermo Fisher primer express software
    The Primer Express Software v3 0 1 allows you to design your own primers and probes using TaqMan and SYBR Green I dye chemistries for gene quantitation and allelic discrimination SNP real time PCR applications Developed specifically for use with our StepOne StepOnePlus 7300 7500 7500 Fast 7900HT ViiA 7 and QuantStudio real time PCR systems Primer Express Software provides customized application specific documents for absolute⁄relative quantitation and allelic discrimination • Enables individual oligonucleotide design for real time PCR applications • Supports assays based on TaqMan and SYBR Green I dye chemistries • Provides design flexibility ease of use and requires minimal optimization • Provides robust assay performance when used in accordance with Rapid Assay Development Guidelines RADG Easy to Learn SoftwareThe Primer Express Software v3 0 1 is flexible easy to learn and to use and requires minimal optimization It includes an algorithm optimized specifically for use with TaqMan based reagents recommended primer⁄probe concentrations and default thermal cycling conditions Primer Express Software also supports assays based on the SYBR Green I dye This minimizes the need for assay optimization and streamlines the design process The software also allows customized designs if the probe and⁄or one or more primers are known Default and adjustable parameters Figure 1 provide the flexibility required for all users regardSimple to UseSimplicity and flexibility are the hallmarks of Primer Express Software v3 0 1 which is fully compatible with all StepOne StepOnePlus 7300 7500 7500 Fast 7900HT ViiA 7 and QuantStudio real time PCR systems Whether your research includes gene quantitation or allelic discrimination studies Primer Express Software belongs in your lab Automated and Manual Primer⁄Probe DesignThe Primer Express Software v3 0 1 will design your primers and probes automatically or you can design them manually For automated design simply choose either TaqMan or TaqMan MGB assays If you know the sequence you need only to import or copy and paste the sequence and quickly view your results Alternatively if you prefer to design the primers and probes manually the procedure is equally simple and straight forward as well Rapid Assay Development GuidelinesThe Primer Express Software v3 0 1 was designed with our Rapid Assay Development Guidelines RADG The RADG are a simple yet comprehensive set of guidelines for use with our real time PCR instruments and reagents Many traditional variables including magnesium chloride concentration and the thermal cycling protocol itself have been standardized which substantially reduces assay development time For Research Use Only Not for use in diagnostics procedures
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    Software:

    Article Title: Identification of Predictive Biomarkers for Lymph Node Involvement in Obese Women With Endometrial Cancer
    Article Snippet: .. The primer sequences were designed using Primer Express™ Software v3.0.1 (ThermoFisher Scientific, USA) (Supplemental material). ..

    Article Title: Molecular Detection of Soil-Transmitted Helminths and Enteric Protozoa Infection in Children and Its Association with Household Water and Sanitation in Manhiça District, Southern Mozambique
    Article Snippet: .. Primer/probes were designed using Primer Express Software v3.0.1 (Applied Biosystems). ..

    Article Title: Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods
    Article Snippet: .. The G. smithogilvyi TaqMan® MGB probes and primer (Table ) were designed using the Primer Express® Software 3.0 (Applied Biosystems, Foster City, CA, USA) on the basis of the same previously described sequences (KR072534). ..

    Article Title: The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule
    Article Snippet: .. Primers for Atp6ap2 were designed using Primer Express software from Applied Biosystems and purchased from Microsynth, Switzerland. ..

    Article Title: From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view
    Article Snippet: .. Primers for the amplification of the CgPWP5 , CgAED2 , CgAWP13 , and CgACT1 cDNA were designed using Primer Express Software (Applied Biosystems) (Supplementary Table ). ..

    Article Title: Virulence of Trypanosoma cruzi Strains Is Related to the Differential Expression of Innate Immune Receptors in the Heart
    Article Snippet: .. The mRNA expression levels were detected by real-time PCR (qPCR) using Fast SYBR Green® Master Mix (Applied Biosystems, USA) according to the instructions of the manufacturer and specific primers , which were obtained by the Primer Express software (Applied Biosystems, USA). ..

    Article Title: Reduced neutrophil elastase inhibitor elafin and elevated transforming growth factor-β1 are linked to inflammatory response in sputum of cystic fibrosis patients with Pseudomonas aeruginosa
    Article Snippet: .. Primers were designed using Primer Express Software v3.0.1 (Thermo Fisher Scientific, Waltham, MA, USA); primer pairs are listed in supplementary table S1. ..

    Article Title: Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate
    Article Snippet: .. The primers were designed based on published gene sequences or by using Primer Express® software package (Applied Biosystems) to create oligonucleotides with similar melting temperatures and minimal self-complementary, and purchased from Eurofins Genomics (Ebersberg, Germany). ..

    Amplification:

    Article Title: From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view
    Article Snippet: .. Primers for the amplification of the CgPWP5 , CgAED2 , CgAWP13 , and CgACT1 cDNA were designed using Primer Express Software (Applied Biosystems) (Supplementary Table ). ..

    Expressing:

    Article Title: Virulence of Trypanosoma cruzi Strains Is Related to the Differential Expression of Innate Immune Receptors in the Heart
    Article Snippet: .. The mRNA expression levels were detected by real-time PCR (qPCR) using Fast SYBR Green® Master Mix (Applied Biosystems, USA) according to the instructions of the manufacturer and specific primers , which were obtained by the Primer Express software (Applied Biosystems, USA). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Virulence of Trypanosoma cruzi Strains Is Related to the Differential Expression of Innate Immune Receptors in the Heart
    Article Snippet: .. The mRNA expression levels were detected by real-time PCR (qPCR) using Fast SYBR Green® Master Mix (Applied Biosystems, USA) according to the instructions of the manufacturer and specific primers , which were obtained by the Primer Express software (Applied Biosystems, USA). ..

    SYBR Green Assay:

    Article Title: Virulence of Trypanosoma cruzi Strains Is Related to the Differential Expression of Innate Immune Receptors in the Heart
    Article Snippet: .. The mRNA expression levels were detected by real-time PCR (qPCR) using Fast SYBR Green® Master Mix (Applied Biosystems, USA) according to the instructions of the manufacturer and specific primers , which were obtained by the Primer Express software (Applied Biosystems, USA). ..

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    Thermo Fisher gene exp ccne1 hs00233356 m1
    Expression analysis of the cyclin E-overexpressing mouse model. A , tail genomic DNA was isolated from founders and their progenies to confirm insertion of the transgene via PCR analysis using human cyclin E-specific primers. DNA from wild-type mouse was used as a negative control.  B , quantitative RT- PCR was applied to verify RNA expression (shown here is  line 11 ). Taqman human cyclin E1 primers were used, and data were normalized to GAPDH via the ΔΔCT method. Murine cyclin E1 expression was also determined, using specific Taqman murine cyclin E1 primers,  p >  0.05.  C ,  left panel ) using purified MK lysates. Human cyclin E ( h-cyclin E ) was detected with anti-human cyclin E antibody. The same amount of protein extract from HeLa cells was used as a positive control. Anti-β-actin was used as loading control.  Right panel , Western blot analysis verifies that transgene expression is specific to MKs. MKs were enriched using the MACS® system as described under “Experimental Procedures.” The rest of the BM fraction (MK depleted-BM) was used to confirm that h-cyclin E is specific to MKs. CD41 antibody was used as MK marker.  D , Western blot analysis using an antibody that detects both human and murine cyclin E shows that total levels of cyclin E are up-regulated in freshly derived cells from transgenic mice and Wt controls. One representative out of three experiments is shown.  h-cyclin E , human cyclin E.  E , protein expression for total cyclin E (shown in  panel D ) was normalized to β-actin and presented as fold change. Quantification was performed by using the NIH ImageJ software (version 1.41o). The data shown represent the averages ± S.D. of three experiments using the Student's  t  test; **,  p
    Gene Exp Ccne1 Hs00233356 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher gene exp ttr hs00174914 m1
    ( A ) Amyloid plaque in the stomach that stains with Congo red and exhibits apple green birefringence ( A i A ii ). The same plaque stains Thioflavin S positive ( A iii ) and is composed of human transthyretin ( A iv ). The area of co-localization of Thioflavin S and <t>TTR</t> labelling appears yellow ( A v ) and morphometric measurements are carried out with the Image J software ( A vi ), ( B ) Morphometric comparison of amyloid deposition in the two backgrounds. Western blots of human non fibrillar TTR in the transgenic animals of the two backgrounds: in the liver ( C ), the serum ( D ) and stomach tissue ( E ).
    Gene Exp Ttr Hs00174914 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp il1b mm00434228 m1
    MyD88-deficient mice exhibit delayed wound healing and decreased inflammatory cytokine expression. A: Wounds were created in Myd88 −/− and control mice. Representative photographs of the wounds of Myd88 −/− mice and controls on days 0 and 3 post injury are shown. The change in wound area was recorded daily by blinded observer and analyzed with NIH ImageJ software (n = 4, repeated 2X). B: Wound myeloid cells CD11b + [CD3 − CD19 − Ly6G − ] were isolated and <t>Il1b</t> and Tnfa expression was quantified using qPCR (n=5). Data are presented as the mean±SEM. Data are representative of 2–3 independent experiments. Data were first analyzed for normal distribution and if data passed normality test, 2-tailed Student t test was used.
    Gene Exp Il1b Mm00434228 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher nt 3 primers
    <t>NT-3</t> expression in RGCs. A , NT-3-like immunoreactivity in the ganglion cell layer of a 16-d-old chick embryo. The cryosection is immunolabeled with the monoclonal NT-3 antibody. Scale bar, 10 μm. B , RT-PCR analysis for NT-3 expression in 1000 purified ( purif ) RGCs, 1000 random retinal cells ( RC ), and 2000 random tectal cells ( TC ). The number of base pairs (400, 500) is indicated. The NT-3 product is 498 bp; the 18 S rRNA product is 488 bp. Note that the NT-3 signal is obtained from purified RGCs and random retinal cells but not from random tectal cells. − RT , Minus reverse transcriptase; 18S , 18 S ribosomal RNA.
    Nt 3 Primers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression analysis of the cyclin E-overexpressing mouse model. A , tail genomic DNA was isolated from founders and their progenies to confirm insertion of the transgene via PCR analysis using human cyclin E-specific primers. DNA from wild-type mouse was used as a negative control.  B , quantitative RT- PCR was applied to verify RNA expression (shown here is  line 11 ). Taqman human cyclin E1 primers were used, and data were normalized to GAPDH via the ΔΔCT method. Murine cyclin E1 expression was also determined, using specific Taqman murine cyclin E1 primers,  p >  0.05.  C ,  left panel ) using purified MK lysates. Human cyclin E ( h-cyclin E ) was detected with anti-human cyclin E antibody. The same amount of protein extract from HeLa cells was used as a positive control. Anti-β-actin was used as loading control.  Right panel , Western blot analysis verifies that transgene expression is specific to MKs. MKs were enriched using the MACS® system as described under “Experimental Procedures.” The rest of the BM fraction (MK depleted-BM) was used to confirm that h-cyclin E is specific to MKs. CD41 antibody was used as MK marker.  D , Western blot analysis using an antibody that detects both human and murine cyclin E shows that total levels of cyclin E are up-regulated in freshly derived cells from transgenic mice and Wt controls. One representative out of three experiments is shown.  h-cyclin E , human cyclin E.  E , protein expression for total cyclin E (shown in  panel D ) was normalized to β-actin and presented as fold change. Quantification was performed by using the NIH ImageJ software (version 1.41o). The data shown represent the averages ± S.D. of three experiments using the Student's  t  test; **,  p

    Journal: The Journal of Biological Chemistry

    Article Title: New Roles for Cyclin E in Megakaryocytic Polyploidization *

    doi: 10.1074/jbc.M110.102145

    Figure Lengend Snippet: Expression analysis of the cyclin E-overexpressing mouse model. A , tail genomic DNA was isolated from founders and their progenies to confirm insertion of the transgene via PCR analysis using human cyclin E-specific primers. DNA from wild-type mouse was used as a negative control. B , quantitative RT- PCR was applied to verify RNA expression (shown here is line 11 ). Taqman human cyclin E1 primers were used, and data were normalized to GAPDH via the ΔΔCT method. Murine cyclin E1 expression was also determined, using specific Taqman murine cyclin E1 primers, p > 0.05. C , left panel ) using purified MK lysates. Human cyclin E ( h-cyclin E ) was detected with anti-human cyclin E antibody. The same amount of protein extract from HeLa cells was used as a positive control. Anti-β-actin was used as loading control. Right panel , Western blot analysis verifies that transgene expression is specific to MKs. MKs were enriched using the MACS® system as described under “Experimental Procedures.” The rest of the BM fraction (MK depleted-BM) was used to confirm that h-cyclin E is specific to MKs. CD41 antibody was used as MK marker. D , Western blot analysis using an antibody that detects both human and murine cyclin E shows that total levels of cyclin E are up-regulated in freshly derived cells from transgenic mice and Wt controls. One representative out of three experiments is shown. h-cyclin E , human cyclin E. E , protein expression for total cyclin E (shown in panel D ) was normalized to β-actin and presented as fold change. Quantification was performed by using the NIH ImageJ software (version 1.41o). The data shown represent the averages ± S.D. of three experiments using the Student's t test; **, p

    Article Snippet: The cDNA was used for quantitative reverse transcriptase PCR, performed using mouse cyclin E1 (Mm01266311_m1 Ccne1) and human cyclin E1 (Hs00233356_m1 Ccne1) TaqMan® gene expression primers and probes (Applied Biosystems).

    Techniques: Expressing, Isolation, Polymerase Chain Reaction, Negative Control, Quantitative RT-PCR, RNA Expression, Purification, Positive Control, Western Blot, Magnetic Cell Separation, Marker, Derivative Assay, Transgenic Assay, Mouse Assay, Software

    ( A ) Amyloid plaque in the stomach that stains with Congo red and exhibits apple green birefringence ( A i A ii ). The same plaque stains Thioflavin S positive ( A iii ) and is composed of human transthyretin ( A iv ). The area of co-localization of Thioflavin S and TTR labelling appears yellow ( A v ) and morphometric measurements are carried out with the Image J software ( A vi ), ( B ) Morphometric comparison of amyloid deposition in the two backgrounds. Western blots of human non fibrillar TTR in the transgenic animals of the two backgrounds: in the liver ( C ), the serum ( D ) and stomach tissue ( E ).

    Journal: Biochemistry and Biophysics Reports

    Article Title: Genetic background modifies amyloidosis in a mouse model of ATTR neuropathy

    doi: 10.1016/j.bbrep.2016.08.005

    Figure Lengend Snippet: ( A ) Amyloid plaque in the stomach that stains with Congo red and exhibits apple green birefringence ( A i A ii ). The same plaque stains Thioflavin S positive ( A iii ) and is composed of human transthyretin ( A iv ). The area of co-localization of Thioflavin S and TTR labelling appears yellow ( A v ) and morphometric measurements are carried out with the Image J software ( A vi ), ( B ) Morphometric comparison of amyloid deposition in the two backgrounds. Western blots of human non fibrillar TTR in the transgenic animals of the two backgrounds: in the liver ( C ), the serum ( D ) and stomach tissue ( E ).

    Article Snippet: 5 µl 2x TaqMan® Gene expression master mix was used with 0.5 µl hTTR primer probe mix (Hs00174914_m1, 4331182) and 2 µl cDNA in a total reaction of 10 µl.

    Techniques: Software, Western Blot, Transgenic Assay

    ( A ) Morphometric comparison for megalin and Western blot comparison for clusterin ( B ) in the two backgrounds, ( C) Intracellular co-localization of TTR with clusterin ( A i -A iv ) and megalin ( A v -A viii ) in stroma stomach cells.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Genetic background modifies amyloidosis in a mouse model of ATTR neuropathy

    doi: 10.1016/j.bbrep.2016.08.005

    Figure Lengend Snippet: ( A ) Morphometric comparison for megalin and Western blot comparison for clusterin ( B ) in the two backgrounds, ( C) Intracellular co-localization of TTR with clusterin ( A i -A iv ) and megalin ( A v -A viii ) in stroma stomach cells.

    Article Snippet: 5 µl 2x TaqMan® Gene expression master mix was used with 0.5 µl hTTR primer probe mix (Hs00174914_m1, 4331182) and 2 µl cDNA in a total reaction of 10 µl.

    Techniques: Western Blot

    ( A ) Real-time PCR results of the number of transgene copies found in the animals of TM129 pure background and TM129/BL6 mixed background, ( B) Reverse transcription real-time PCR results from the liver tissue of TM129 pure background and TM129/BL6 mixed background animals measuring the amount of human TTR RNA expressed in liver, ( C) Western blot of TM129 stomach samples from different mice and a single wild type 129 animal for hTTR and GAPDH. The strong TTR band seen consists of monomers.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Genetic background modifies amyloidosis in a mouse model of ATTR neuropathy

    doi: 10.1016/j.bbrep.2016.08.005

    Figure Lengend Snippet: ( A ) Real-time PCR results of the number of transgene copies found in the animals of TM129 pure background and TM129/BL6 mixed background, ( B) Reverse transcription real-time PCR results from the liver tissue of TM129 pure background and TM129/BL6 mixed background animals measuring the amount of human TTR RNA expressed in liver, ( C) Western blot of TM129 stomach samples from different mice and a single wild type 129 animal for hTTR and GAPDH. The strong TTR band seen consists of monomers.

    Article Snippet: 5 µl 2x TaqMan® Gene expression master mix was used with 0.5 µl hTTR primer probe mix (Hs00174914_m1, 4331182) and 2 µl cDNA in a total reaction of 10 µl.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Mouse Assay

    MyD88-deficient mice exhibit delayed wound healing and decreased inflammatory cytokine expression. A: Wounds were created in Myd88 −/− and control mice. Representative photographs of the wounds of Myd88 −/− mice and controls on days 0 and 3 post injury are shown. The change in wound area was recorded daily by blinded observer and analyzed with NIH ImageJ software (n = 4, repeated 2X). B: Wound myeloid cells CD11b + [CD3 − CD19 − Ly6G − ] were isolated and Il1b and Tnfa expression was quantified using qPCR (n=5). Data are presented as the mean±SEM. Data are representative of 2–3 independent experiments. Data were first analyzed for normal distribution and if data passed normality test, 2-tailed Student t test was used.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Histone Methylation Directs Myeloid Toll-like Receptor 4 Expression and Regulates Wound Healing Following Cutaneous Tissue Injury

    doi: 10.4049/jimmunol.1801258

    Figure Lengend Snippet: MyD88-deficient mice exhibit delayed wound healing and decreased inflammatory cytokine expression. A: Wounds were created in Myd88 −/− and control mice. Representative photographs of the wounds of Myd88 −/− mice and controls on days 0 and 3 post injury are shown. The change in wound area was recorded daily by blinded observer and analyzed with NIH ImageJ software (n = 4, repeated 2X). B: Wound myeloid cells CD11b + [CD3 − CD19 − Ly6G − ] were isolated and Il1b and Tnfa expression was quantified using qPCR (n=5). Data are presented as the mean±SEM. Data are representative of 2–3 independent experiments. Data were first analyzed for normal distribution and if data passed normality test, 2-tailed Student t test was used.

    Article Snippet: Primers for Il1b (Mm00434228_m10), Tnfa (Mm00443258_m1), Mll1 (Mm01179235_m1), Cd44 (Mm0 1277164-m1), Tlr4 (Mm00445273_m1) were purchased (Applied Biosystems).

    Techniques: Mouse Assay, Expressing, Software, Isolation, Real-time Polymerase Chain Reaction

    Myeloid-specific TLR4 was sufficient to rescue wound healing in Tlr4 −/− mice. A: Myeloid depletion of Tlr54 was examined by qPCR in MACS splenic myeloid cells CD11b+[CD3 − CD19 − Ly6G − ] from Tlr4 f/f Lyz2 Cre+ mice and littermate controls ( Tlr4 f/f Lyz2 Cre- ) (n= 10). B: Wounds were created by 4-mm punch biopsy on the backs of Tlr4 f/f Lyz2 Cre+ mice and littermate control mice. The change in wound area was recorded daily with ImageJ software until complete healing was observed (n=10). C: CD3 − CD11c − CD19 − Ly6G − NK1.1 − CD11b + single cell suspensions were isolated from Tlr4 −/− and Tlr4 +/+ spleens expressing CD45.1 by MACS. Cells (1 × 10 6 ) were injected intravenously in wounded (day 1) Tlr4 −/− mice expressing CD45.2. Tracking of CD45.1 cells to the wounds was identified by the previously mentioned gating strategy and quantified as shown above. D: Wound myeloid cells CD11b + [CD3 − CD19 − Ly6G − ] were isolated and Il1b and Tnfa expression was quantified using qPCR on day 4 post-wound (day 3 post-transfer) (n=3). E: Wound closure was measured in recipient mice daily by blinded observers with ImageJ software (n = 15) (n=15). B: Representative images are shown from Tlr4 −/− → Tlr4 −/− and Tlr4 +/+ → Tlr4 −/− mice on days 0 and 3. Data are presented as the mean±SEM. Data are representative of 2–3 independent experiments. Data were first analyzed for normal distribution and if data passed normality test, 2-tailed Student t test was used.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Histone Methylation Directs Myeloid Toll-like Receptor 4 Expression and Regulates Wound Healing Following Cutaneous Tissue Injury

    doi: 10.4049/jimmunol.1801258

    Figure Lengend Snippet: Myeloid-specific TLR4 was sufficient to rescue wound healing in Tlr4 −/− mice. A: Myeloid depletion of Tlr54 was examined by qPCR in MACS splenic myeloid cells CD11b+[CD3 − CD19 − Ly6G − ] from Tlr4 f/f Lyz2 Cre+ mice and littermate controls ( Tlr4 f/f Lyz2 Cre- ) (n= 10). B: Wounds were created by 4-mm punch biopsy on the backs of Tlr4 f/f Lyz2 Cre+ mice and littermate control mice. The change in wound area was recorded daily with ImageJ software until complete healing was observed (n=10). C: CD3 − CD11c − CD19 − Ly6G − NK1.1 − CD11b + single cell suspensions were isolated from Tlr4 −/− and Tlr4 +/+ spleens expressing CD45.1 by MACS. Cells (1 × 10 6 ) were injected intravenously in wounded (day 1) Tlr4 −/− mice expressing CD45.2. Tracking of CD45.1 cells to the wounds was identified by the previously mentioned gating strategy and quantified as shown above. D: Wound myeloid cells CD11b + [CD3 − CD19 − Ly6G − ] were isolated and Il1b and Tnfa expression was quantified using qPCR on day 4 post-wound (day 3 post-transfer) (n=3). E: Wound closure was measured in recipient mice daily by blinded observers with ImageJ software (n = 15) (n=15). B: Representative images are shown from Tlr4 −/− → Tlr4 −/− and Tlr4 +/+ → Tlr4 −/− mice on days 0 and 3. Data are presented as the mean±SEM. Data are representative of 2–3 independent experiments. Data were first analyzed for normal distribution and if data passed normality test, 2-tailed Student t test was used.

    Article Snippet: Primers for Il1b (Mm00434228_m10), Tnfa (Mm00443258_m1), Mll1 (Mm01179235_m1), Cd44 (Mm0 1277164-m1), Tlr4 (Mm00445273_m1) were purchased (Applied Biosystems).

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Magnetic Cell Separation, Software, Isolation, Expressing, Injection

    Decreased inflammatory cytokine expression in TLR4-deficient macrophages in vitro and in vivo . A: Bone marrow derived macrophages (BMDMs) harvested from Tlr4 −/− mice and controls were stimulated with LPS (100 ng/mL) for 2 hours after which they were collected for analysis. Il1b and Tnfa gene expression was quantified by qPCR (n = 5). B: Wound myeloid cells CD11b + [CD3 − CD19 − Ly6G − ] were isolated and Il1b and Tnfa expression was quantified using qPCR (n=5). C: Tlr4 −/− and control wound cell isolates were processed for intracellular flow cytometry. The gating strategy used for intracellular flow cytometry selecting live, lineage − , Ly6G − , CD11b + cells is shown. D: Flow cytometry quantification of IL1β and TNFα in wounds (n = 10). Data are presented as the mean±SEM. Data are representative of 2–3 independent experiments. Data were first analyzed for normal distribution and if data passed normality test, 2-tailed Student t test was used. FMO, fluorescence minus one; FSC, forward scatter; FSC-A, forward scatter area; FSC-H, forward scatter height; SSC, side scatter.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Histone Methylation Directs Myeloid Toll-like Receptor 4 Expression and Regulates Wound Healing Following Cutaneous Tissue Injury

    doi: 10.4049/jimmunol.1801258

    Figure Lengend Snippet: Decreased inflammatory cytokine expression in TLR4-deficient macrophages in vitro and in vivo . A: Bone marrow derived macrophages (BMDMs) harvested from Tlr4 −/− mice and controls were stimulated with LPS (100 ng/mL) for 2 hours after which they were collected for analysis. Il1b and Tnfa gene expression was quantified by qPCR (n = 5). B: Wound myeloid cells CD11b + [CD3 − CD19 − Ly6G − ] were isolated and Il1b and Tnfa expression was quantified using qPCR (n=5). C: Tlr4 −/− and control wound cell isolates were processed for intracellular flow cytometry. The gating strategy used for intracellular flow cytometry selecting live, lineage − , Ly6G − , CD11b + cells is shown. D: Flow cytometry quantification of IL1β and TNFα in wounds (n = 10). Data are presented as the mean±SEM. Data are representative of 2–3 independent experiments. Data were first analyzed for normal distribution and if data passed normality test, 2-tailed Student t test was used. FMO, fluorescence minus one; FSC, forward scatter; FSC-A, forward scatter area; FSC-H, forward scatter height; SSC, side scatter.

    Article Snippet: Primers for Il1b (Mm00434228_m10), Tnfa (Mm00443258_m1), Mll1 (Mm01179235_m1), Cd44 (Mm0 1277164-m1), Tlr4 (Mm00445273_m1) were purchased (Applied Biosystems).

    Techniques: Expressing, In Vitro, In Vivo, Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation, Flow Cytometry, Fluorescence

    NT-3 expression in RGCs. A , NT-3-like immunoreactivity in the ganglion cell layer of a 16-d-old chick embryo. The cryosection is immunolabeled with the monoclonal NT-3 antibody. Scale bar, 10 μm. B , RT-PCR analysis for NT-3 expression in 1000 purified ( purif ) RGCs, 1000 random retinal cells ( RC ), and 2000 random tectal cells ( TC ). The number of base pairs (400, 500) is indicated. The NT-3 product is 498 bp; the 18 S rRNA product is 488 bp. Note that the NT-3 signal is obtained from purified RGCs and random retinal cells but not from random tectal cells. − RT , Minus reverse transcriptase; 18S , 18 S ribosomal RNA.

    Journal: The Journal of Neuroscience

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Figure Lengend Snippet: NT-3 expression in RGCs. A , NT-3-like immunoreactivity in the ganglion cell layer of a 16-d-old chick embryo. The cryosection is immunolabeled with the monoclonal NT-3 antibody. Scale bar, 10 μm. B , RT-PCR analysis for NT-3 expression in 1000 purified ( purif ) RGCs, 1000 random retinal cells ( RC ), and 2000 random tectal cells ( TC ). The number of base pairs (400, 500) is indicated. The NT-3 product is 498 bp; the 18 S rRNA product is 488 bp. Note that the NT-3 signal is obtained from purified RGCs and random retinal cells but not from random tectal cells. − RT , Minus reverse transcriptase; 18S , 18 S ribosomal RNA.

    Article Snippet: NT-3 primers (Life Technologies) were designed by using Mac-Vector software, and 18 S rRNA primers were purchased from commercial sources (Ambion, Inc.).

    Techniques: Expressing, Immunolabeling, Reverse Transcription Polymerase Chain Reaction, Purification

    Sections through the retina of a 16-d-old chick embryo hybridized with a probe for NT-3 ( A, B ) or immunolabeled for NT-3 protein ( C, D ). A , Section hybridized with the antisense probe for NT-3. Note the label in some cells ( arrowhead ) within the GCL. B , Adjacent section hybridized with a sense control probe. C , Immunolabeled section through the normal (control) retina. Note that the INL is labeled differentially; the inner half is labeled more intensely than the outer half. D , Section through the retina of the same embryo in which pertussis toxin ( PTX ) was injected into the eye. Note that PTX changed the pattern of NT-3 distribution that became homogeneous throughout the INL, but PTX treatment did not consistently change the intensity or distribution of NT-3 label in the GCL. Scale bars: A, B, 10 μm; C, D, 20 μm.

    Journal: The Journal of Neuroscience

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Figure Lengend Snippet: Sections through the retina of a 16-d-old chick embryo hybridized with a probe for NT-3 ( A, B ) or immunolabeled for NT-3 protein ( C, D ). A , Section hybridized with the antisense probe for NT-3. Note the label in some cells ( arrowhead ) within the GCL. B , Adjacent section hybridized with a sense control probe. C , Immunolabeled section through the normal (control) retina. Note that the INL is labeled differentially; the inner half is labeled more intensely than the outer half. D , Section through the retina of the same embryo in which pertussis toxin ( PTX ) was injected into the eye. Note that PTX changed the pattern of NT-3 distribution that became homogeneous throughout the INL, but PTX treatment did not consistently change the intensity or distribution of NT-3 label in the GCL. Scale bars: A, B, 10 μm; C, D, 20 μm.

    Article Snippet: NT-3 primers (Life Technologies) were designed by using Mac-Vector software, and 18 S rRNA primers were purchased from commercial sources (Ambion, Inc.).

    Techniques: Immunolabeling, Labeling, Injection

    Sections through the superficial layers of the optic tectum of 16-d-old chick embryos showing the distribution of exogenous and endogenous ( endog .) NT-3. A , Distribution of radiolabeled NT-3 in the optic tectum after injection in the eye and anterograde transport by retinal ganglion cell axons is shown. Dark-field image of a paraffin section processed for autoradiography. B, Normal tissue section immunolabeled with pAB to NT-3 shows a strong band of neuropil label in the same layers, the SO, and the SGFSa–d as well as cellular label in the SGC. Free-floating cryosection. The same pattern was seen with mAB to NT-3 (shown in D ). C , No label was seen in control sections in which the primary antibody was replaced by irrelevant IgG. Free-floating cryosection. The layers SGFSg ( g ) and SGC are indicated. D , Section through the ipsilateral (control) optic tectum 48 hr after the intraocular injection of colchicine and immunolabeled with mAB to NT-3 is shown. Free-floating section. Note a normal band of NT-3 immunoreactivity. E, Same section shown in D but from the contralateral optic tectum. Note that intraocular colchicine ( COL ) largely reduced the NT-3 label in the SO and SGFSa–d, demonstrating that NT-3-like immunoreactivity was derived from the retina and was present in retinotectal axons. F , Fluorescent label in the SO and SGFSa–d layers of the contralateral tectum after injection of DiI into the eye is shown. Cryosection. Scale bars: A, B–E, F , 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Figure Lengend Snippet: Sections through the superficial layers of the optic tectum of 16-d-old chick embryos showing the distribution of exogenous and endogenous ( endog .) NT-3. A , Distribution of radiolabeled NT-3 in the optic tectum after injection in the eye and anterograde transport by retinal ganglion cell axons is shown. Dark-field image of a paraffin section processed for autoradiography. B, Normal tissue section immunolabeled with pAB to NT-3 shows a strong band of neuropil label in the same layers, the SO, and the SGFSa–d as well as cellular label in the SGC. Free-floating cryosection. The same pattern was seen with mAB to NT-3 (shown in D ). C , No label was seen in control sections in which the primary antibody was replaced by irrelevant IgG. Free-floating cryosection. The layers SGFSg ( g ) and SGC are indicated. D , Section through the ipsilateral (control) optic tectum 48 hr after the intraocular injection of colchicine and immunolabeled with mAB to NT-3 is shown. Free-floating section. Note a normal band of NT-3 immunoreactivity. E, Same section shown in D but from the contralateral optic tectum. Note that intraocular colchicine ( COL ) largely reduced the NT-3 label in the SO and SGFSa–d, demonstrating that NT-3-like immunoreactivity was derived from the retina and was present in retinotectal axons. F , Fluorescent label in the SO and SGFSa–d layers of the contralateral tectum after injection of DiI into the eye is shown. Cryosection. Scale bars: A, B–E, F , 100 μm.

    Article Snippet: NT-3 primers (Life Technologies) were designed by using Mac-Vector software, and 18 S rRNA primers were purchased from commercial sources (Ambion, Inc.).

    Techniques: Injection, Paraffin Section, Autoradiography, Immunolabeling, Derivative Assay

    Synopsis of NT-3 immunolabel and neurotrophin receptor immunolabel in the superficial layers of the optic tectum in 16-d-old chick embryos and their sources from within the tectum or retinal projection. Shaded ( gray ) areas indicate neuropil label, graded from light (low levels) to dark (high levels). Labeled cell bodies are indicated by dots or profiles (when dendritic details are labeled). +MON , Monensin added in the eye (to reveal nonretinal source of label; intraocular monensin prevents anterograde axonal transport and causes degeneration of RGC axons and axon terminals). The right - hand panel depicts the fiber course of retinal ganglion cell axons in the SO and the SGFS and the morphology of tectal neurons in sublayer SGFSi with ascending dendrites into SGFSg, d, and c.

    Journal: The Journal of Neuroscience

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Figure Lengend Snippet: Synopsis of NT-3 immunolabel and neurotrophin receptor immunolabel in the superficial layers of the optic tectum in 16-d-old chick embryos and their sources from within the tectum or retinal projection. Shaded ( gray ) areas indicate neuropil label, graded from light (low levels) to dark (high levels). Labeled cell bodies are indicated by dots or profiles (when dendritic details are labeled). +MON , Monensin added in the eye (to reveal nonretinal source of label; intraocular monensin prevents anterograde axonal transport and causes degeneration of RGC axons and axon terminals). The right - hand panel depicts the fiber course of retinal ganglion cell axons in the SO and the SGFS and the morphology of tectal neurons in sublayer SGFSi with ascending dendrites into SGFSg, d, and c.

    Article Snippet: NT-3 primers (Life Technologies) were designed by using Mac-Vector software, and 18 S rRNA primers were purchased from commercial sources (Ambion, Inc.).

    Techniques: Immunolabeling, Labeling

    Specificity and sensitivity of the monoclonal NT-3 antibody (mAB 1D53B2). A , Dot blot of NT-3 mAB showing its sensitivity and specificity for NT-3 compared with NGF, BDNF, and NT-4. The antibody detected

    Journal: The Journal of Neuroscience

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Figure Lengend Snippet: Specificity and sensitivity of the monoclonal NT-3 antibody (mAB 1D53B2). A , Dot blot of NT-3 mAB showing its sensitivity and specificity for NT-3 compared with NGF, BDNF, and NT-4. The antibody detected

    Article Snippet: NT-3 primers (Life Technologies) were designed by using Mac-Vector software, and 18 S rRNA primers were purchased from commercial sources (Ambion, Inc.).

    Techniques: Dot Blot

    Effects of intraocular monensin, colchicine, and NT-3 antibodies on retinotectal projections in chick embryos. A , DiI application to the fixed retinotectal projection shows DiI label in the SO and SGFSd ( d, arrowheads ). B , Pretreatment with monensin ( +Mon ) in the eye abolishes DiI label in SGFSd and results in diffuse DiI label in the SO. C , The thickness ( arrow ) of the SO and SGFSa–f in the normal tectum [layer SGFSg is indicated ( g )] is shown. D , Pretreatment of the eye with colchicine ( +Col ) reduces the thickness ( arrow ) of the SO and SGFSa–f. E , Quantification of changes in the thickness of the SO + SGFSa–f tectal layers (ipsilateral vehicle control side = 100%) is shown. Note that axotomy, monensin, and colchicine cause a similar reduction in the thickness of the retinorecipient tectal layers, at least in part because of degeneration of retinotectal fibers and terminals. Error bars indicate SEM. The number of independent experiments ( n ) is indicated. a-NT3 , NT-3 antibody; Axo , axotomy; IgG , normal IgG control; Veh , vehicle. Scale bars: A, B ; C, D , 50 μm.

    Journal: The Journal of Neuroscience

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Figure Lengend Snippet: Effects of intraocular monensin, colchicine, and NT-3 antibodies on retinotectal projections in chick embryos. A , DiI application to the fixed retinotectal projection shows DiI label in the SO and SGFSd ( d, arrowheads ). B , Pretreatment with monensin ( +Mon ) in the eye abolishes DiI label in SGFSd and results in diffuse DiI label in the SO. C , The thickness ( arrow ) of the SO and SGFSa–f in the normal tectum [layer SGFSg is indicated ( g )] is shown. D , Pretreatment of the eye with colchicine ( +Col ) reduces the thickness ( arrow ) of the SO and SGFSa–f. E , Quantification of changes in the thickness of the SO + SGFSa–f tectal layers (ipsilateral vehicle control side = 100%) is shown. Note that axotomy, monensin, and colchicine cause a similar reduction in the thickness of the retinorecipient tectal layers, at least in part because of degeneration of retinotectal fibers and terminals. Error bars indicate SEM. The number of independent experiments ( n ) is indicated. a-NT3 , NT-3 antibody; Axo , axotomy; IgG , normal IgG control; Veh , vehicle. Scale bars: A, B ; C, D , 50 μm.

    Article Snippet: NT-3 primers (Life Technologies) were designed by using Mac-Vector software, and 18 S rRNA primers were purchased from commercial sources (Ambion, Inc.).

    Techniques:

    Effects of the blocking NT-3 mAB in the eye on the distribution of NT-3 and cell death in the tectum. A , Dose–response curve of the effect of blocking mAB to NT-3 in the eye on the transport of radio-iodinated NT-3 from the eye to the midbrain in 15- to 16-d-old chick embryos. mAB (0.4 μg ) blocks ∼60% of the transport, 2 μg blocks ∼90%, and 20 μg blocks ∼95%. Each data point with error bars (SEM) is the average of three to eight experiments. Dashed line, Control antibody. B , C , Sections through the optic tectum of a 16-d-old chick embryo immunolabeled for NT-3 after daily injections of 20 μg of NT-3 mAB in one eye. Note the persistence of NT-3 label in the retinorecipient neuropil of layers SGFSa–d in the antibody-treated retinotectal projection ( C ) compared with the ipsilateral control side ( B ). The experimental tectum showed an increase in the number of NT-3-labeled cell bodies in SGFSg–i. D , Example of a pyknotic cell ( arrowhead ) in the SGC of a 16-d-old chick embryo. E , Quantification of pyknotic neuronal profiles in the optic tectum after injections of 2, 22, or 3 × 22 μg of NT-3 mAB ( a-NT ) in the eye or of irrelevant, control antibody ( CO ) in the eye. Bar graphs show a significant effect ( p ≤ 0.05, t test) of NT-3 mAB in the eye on the cell death of neurons in the SGC of the contralateral optic tectum only after multiple high doses over 4 d. The number of independent experiments is indicated on each vertical bar . Error bars indicate SEM. These data are consistent with the hypothesis that RGCs export primarily their own NT-3 to the optic tectum so that depletion of extrinsic sources becomes relevant only after 3–4 d. Scale bars: B, C, 100 μm; D , 10 μm.

    Journal: The Journal of Neuroscience

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Figure Lengend Snippet: Effects of the blocking NT-3 mAB in the eye on the distribution of NT-3 and cell death in the tectum. A , Dose–response curve of the effect of blocking mAB to NT-3 in the eye on the transport of radio-iodinated NT-3 from the eye to the midbrain in 15- to 16-d-old chick embryos. mAB (0.4 μg ) blocks ∼60% of the transport, 2 μg blocks ∼90%, and 20 μg blocks ∼95%. Each data point with error bars (SEM) is the average of three to eight experiments. Dashed line, Control antibody. B , C , Sections through the optic tectum of a 16-d-old chick embryo immunolabeled for NT-3 after daily injections of 20 μg of NT-3 mAB in one eye. Note the persistence of NT-3 label in the retinorecipient neuropil of layers SGFSa–d in the antibody-treated retinotectal projection ( C ) compared with the ipsilateral control side ( B ). The experimental tectum showed an increase in the number of NT-3-labeled cell bodies in SGFSg–i. D , Example of a pyknotic cell ( arrowhead ) in the SGC of a 16-d-old chick embryo. E , Quantification of pyknotic neuronal profiles in the optic tectum after injections of 2, 22, or 3 × 22 μg of NT-3 mAB ( a-NT ) in the eye or of irrelevant, control antibody ( CO ) in the eye. Bar graphs show a significant effect ( p ≤ 0.05, t test) of NT-3 mAB in the eye on the cell death of neurons in the SGC of the contralateral optic tectum only after multiple high doses over 4 d. The number of independent experiments is indicated on each vertical bar . Error bars indicate SEM. These data are consistent with the hypothesis that RGCs export primarily their own NT-3 to the optic tectum so that depletion of extrinsic sources becomes relevant only after 3–4 d. Scale bars: B, C, 100 μm; D , 10 μm.

    Article Snippet: NT-3 primers (Life Technologies) were designed by using Mac-Vector software, and 18 S rRNA primers were purchased from commercial sources (Ambion, Inc.).

    Techniques: Blocking Assay, Immunolabeling, Labeling