wfa  (Vector Laboratories)


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    Name:
    Fluorescein labeled Wisteria Floribunda Lectin WFA WFL
    Description:
    Wisteria floribunda lectin WFA WFL The binding specificity of Wisteria floribunda lectin WFL is not completely clear but this lectin appears to preferentially bind carbohydrate structures terminating in N acetylgalactosamine linked α or β to the 3 or 6 position of galactose This lectin has been used to fractionate lymphocyte populations and although not mitogenic elicits the production of lymphokines from murine splenocytes Fluorescein labeled WFL has an appropriate number of fluorochromes bound to provide the optimum staining characteristics for this lectin This conjugate is supplied essentially free of unconjugated fluorochromes The excitation maximum is at 495 nm and the emission maximum is at 515 nm Accompanying each fluorescent lectin is an analysis data sheet summarizing the results of our quality control tests and providing pertinent information on the product All of these reagents are supplied as solutions perserved with sodium azide Inhibiting Eluting Sugar 200 mM N acetylgalactosamine
    Catalog Number:
    fl-1351
    Price:
    None
    Category:
    Proteins
    Size:
    2 mg
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    Structured Review

    Vector Laboratories wfa
    Fluorescein labeled Wisteria Floribunda Lectin WFA WFL
    Wisteria floribunda lectin WFA WFL The binding specificity of Wisteria floribunda lectin WFL is not completely clear but this lectin appears to preferentially bind carbohydrate structures terminating in N acetylgalactosamine linked α or β to the 3 or 6 position of galactose This lectin has been used to fractionate lymphocyte populations and although not mitogenic elicits the production of lymphokines from murine splenocytes Fluorescein labeled WFL has an appropriate number of fluorochromes bound to provide the optimum staining characteristics for this lectin This conjugate is supplied essentially free of unconjugated fluorochromes The excitation maximum is at 495 nm and the emission maximum is at 515 nm Accompanying each fluorescent lectin is an analysis data sheet summarizing the results of our quality control tests and providing pertinent information on the product All of these reagents are supplied as solutions perserved with sodium azide Inhibiting Eluting Sugar 200 mM N acetylgalactosamine
    https://www.bioz.com/result/wfa/product/Vector Laboratories
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    wfa - by Bioz Stars, 2021-03
    93/100 stars

    Images

    1) Product Images from "Activity-Dependent Dysfunction in Visual and Olfactory Sensory Systems in Mouse Models of Down Syndrome"

    Article Title: Activity-Dependent Dysfunction in Visual and Olfactory Sensory Systems in Mouse Models of Down Syndrome

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1045-17.2017

    Histologic assessment of neuronal populations in the Ts65Dn visual cortex. A–O , Representative immunostains ( A–L ) and WFA staining ( M–O ) of nontrisomic littermate ( A , D , G , J , M ) and Ts65Dn ( B , E , H , K , N ) visual cortex. No significant differences were observed in the density of NeuN + ( A–C ), Gad67 + ( D–F ), parvalbumin + ( G–I ), somatostatin + ( J–L ), or WFA-stained neurons in Ts65Dn and nontrisomic visual cortex. C , F , I , L , O , Quantitation of cell counts in nontrisomic (black) and Ts65Dn (red) cortex. Scale bar, 500 μm. Error bars denote SD. n = 6 per group.
    Figure Legend Snippet: Histologic assessment of neuronal populations in the Ts65Dn visual cortex. A–O , Representative immunostains ( A–L ) and WFA staining ( M–O ) of nontrisomic littermate ( A , D , G , J , M ) and Ts65Dn ( B , E , H , K , N ) visual cortex. No significant differences were observed in the density of NeuN + ( A–C ), Gad67 + ( D–F ), parvalbumin + ( G–I ), somatostatin + ( J–L ), or WFA-stained neurons in Ts65Dn and nontrisomic visual cortex. C , F , I , L , O , Quantitation of cell counts in nontrisomic (black) and Ts65Dn (red) cortex. Scale bar, 500 μm. Error bars denote SD. n = 6 per group.

    Techniques Used: Staining, Quantitation Assay

    Related Articles

    Staining:

    Article Title: N-Acetylgalactosamine Positive Perineuronal Nets in the Saccade-Related-Part of the Cerebellar Fastigial Nucleus Do Not Maintain Saccade Gain
    Article Snippet: We mounted sections on charged slides, dehydrated them with increasing concentrations of ethanol, stained them with toluidine blue at pH 4.1 for ∼45 seconds, and then rehydrated them. .. We stained another set for intact perineuronal nets with wisteria floribunda agglutinin (WFA) conjugated with fluorescein (1∶500, Vector Labs FL-1351) using standard immunohistochemical techniques . ..

    Article Title: Activity-Dependent Dysfunction in Visual and Olfactory Sensory Systems in Mouse Models of Down Syndrome
    Article Snippet: .. Sections were stained by overnight incubation with guinea pig anti-Arc (catalog #156 005, Synaptic Systems; 1:1000), mouse anti-NeuN (MAB377, Millipore; 1:1000), mouse anti-parvalbumin (P3088, Sigma-Aldrich; 1:4000), rabbit anti-somatostatin (ab64053, Abcam; 1:1000), and mouse anti-Gad67 (MAB5406, Millipore; 1:1000) antibodies and/or WFA (FL-1351, Vector Laboratories; 1:1000) after 10 min postfixation with 4% PFA, permeabilization with 0.3% Tween 20, and blocking with 10% goat serum. .. WFA-stained slides were coverslipped using Slowfade mounting medium without glycerol (S-2828, Thermo Fisher Scientific).

    Article Title: Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms
    Article Snippet: .. After digestion, biofilms were washed, and Pel was then stained with fluorescein-labeled WFL lectin (100 μg/ml; Vector Laboratories) for 15 min. Stained biofilms were washed before visualization on a Zeiss LSM 510 confocal laser scanning microscope. .. Image analysis was conducted using the Velocity software (Improvision).

    Article Title: A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity
    Article Snippet: Coronal brain sections through the prefrontal cortex (PFC; +4.0 through +3.6 from bregma; Paxinos & Watson, 2007) and dorsal hippocampus (−2.0 through −4.0 from bregma; Paxinos & Watson, 2007) were cut at 30 µm using a freezing microtome. .. WFA staining was performed by washing free-floating sections three times for 5 min in 1×-PBS, the tissue was placed in 3% goat blocking serum (Vector Laboratories) for 1 h and was then incubated overnight at 4°C on a shaker table with fluorescein-conjugated WFA (1:500, Vector Laboratories) in 1×-PBS containing 2% goat serum. .. The tissue was washed three times for 10 min each time in 1×-PBS and mounted onto Frost plus slides in diluted 1×-PBS (30:200) with 0.24% Triton X-100 (Sigma-Aldrich).

    Immunohistochemistry:

    Article Title: N-Acetylgalactosamine Positive Perineuronal Nets in the Saccade-Related-Part of the Cerebellar Fastigial Nucleus Do Not Maintain Saccade Gain
    Article Snippet: We mounted sections on charged slides, dehydrated them with increasing concentrations of ethanol, stained them with toluidine blue at pH 4.1 for ∼45 seconds, and then rehydrated them. .. We stained another set for intact perineuronal nets with wisteria floribunda agglutinin (WFA) conjugated with fluorescein (1∶500, Vector Labs FL-1351) using standard immunohistochemical techniques . ..

    Incubation:

    Article Title: Precocious deposition of perineuronal nets on Parvalbumin inhibitory neurons transplanted into adult visual cortex
    Article Snippet: .. They were then incubated for two hours in 594 goat-anti-rabbit IgG (1:1000, Invitrogen) for VGAT cells, 647 goat-anti-mouse IgG1 (1:1000, Invitrogen) for PV cells, and Fluorescein labeled Wisteria Floribunda Agglutinin (WFA), Vector Labs) for PNN+ cells. .. The stained tissue was mounted on glass slides with Fluoroshield with DAPI.

    Article Title: Activity-Dependent Dysfunction in Visual and Olfactory Sensory Systems in Mouse Models of Down Syndrome
    Article Snippet: .. Sections were stained by overnight incubation with guinea pig anti-Arc (catalog #156 005, Synaptic Systems; 1:1000), mouse anti-NeuN (MAB377, Millipore; 1:1000), mouse anti-parvalbumin (P3088, Sigma-Aldrich; 1:4000), rabbit anti-somatostatin (ab64053, Abcam; 1:1000), and mouse anti-Gad67 (MAB5406, Millipore; 1:1000) antibodies and/or WFA (FL-1351, Vector Laboratories; 1:1000) after 10 min postfixation with 4% PFA, permeabilization with 0.3% Tween 20, and blocking with 10% goat serum. .. WFA-stained slides were coverslipped using Slowfade mounting medium without glycerol (S-2828, Thermo Fisher Scientific).

    Article Title: Venlafaxine Stimulates an MMP-9-Dependent Increase in Excitatory/Inhibitory Balance in a Stress Model of Depression
    Article Snippet: Sections were washed 2 or 3 times with 1× PBS, then permeabilized with 1× PBS containing 0.1% Triton X-100, blocked with 10% normal goat serum, and incubated with anti-PV (1:500, Sigma Millipore, P3088) overnight at 4°C. .. Following subsequent washes and incubation with a fluorescent secondary antibody for PV immunostaining and fluorescein-labeled Wisteria floribunda lectin (WFA) (1:1000, Vector Laboratories, FL-1351) for 2 h at room temperature, sections were washed several times with 1× PBS, counterstained with DAPI, and mounted with Hydromount (National Diagnostics, HS-106) before being allowed to dry several days at 4°C. .. For PV and PNN cell quantification, images were acquired using a Leica SP8 laser scanning confocal microscope with an oil immersion, 209 objective with 0.40 numerical aperture.

    Article Title: A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity
    Article Snippet: Coronal brain sections through the prefrontal cortex (PFC; +4.0 through +3.6 from bregma; Paxinos & Watson, 2007) and dorsal hippocampus (−2.0 through −4.0 from bregma; Paxinos & Watson, 2007) were cut at 30 µm using a freezing microtome. .. WFA staining was performed by washing free-floating sections three times for 5 min in 1×-PBS, the tissue was placed in 3% goat blocking serum (Vector Laboratories) for 1 h and was then incubated overnight at 4°C on a shaker table with fluorescein-conjugated WFA (1:500, Vector Laboratories) in 1×-PBS containing 2% goat serum. .. The tissue was washed three times for 10 min each time in 1×-PBS and mounted onto Frost plus slides in diluted 1×-PBS (30:200) with 0.24% Triton X-100 (Sigma-Aldrich).

    Labeling:

    Article Title: Precocious deposition of perineuronal nets on Parvalbumin inhibitory neurons transplanted into adult visual cortex
    Article Snippet: .. They were then incubated for two hours in 594 goat-anti-rabbit IgG (1:1000, Invitrogen) for VGAT cells, 647 goat-anti-mouse IgG1 (1:1000, Invitrogen) for PV cells, and Fluorescein labeled Wisteria Floribunda Agglutinin (WFA), Vector Labs) for PNN+ cells. .. The stained tissue was mounted on glass slides with Fluoroshield with DAPI.

    Blocking Assay:

    Article Title: Activity-Dependent Dysfunction in Visual and Olfactory Sensory Systems in Mouse Models of Down Syndrome
    Article Snippet: .. Sections were stained by overnight incubation with guinea pig anti-Arc (catalog #156 005, Synaptic Systems; 1:1000), mouse anti-NeuN (MAB377, Millipore; 1:1000), mouse anti-parvalbumin (P3088, Sigma-Aldrich; 1:4000), rabbit anti-somatostatin (ab64053, Abcam; 1:1000), and mouse anti-Gad67 (MAB5406, Millipore; 1:1000) antibodies and/or WFA (FL-1351, Vector Laboratories; 1:1000) after 10 min postfixation with 4% PFA, permeabilization with 0.3% Tween 20, and blocking with 10% goat serum. .. WFA-stained slides were coverslipped using Slowfade mounting medium without glycerol (S-2828, Thermo Fisher Scientific).

    Article Title: A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity
    Article Snippet: Coronal brain sections through the prefrontal cortex (PFC; +4.0 through +3.6 from bregma; Paxinos & Watson, 2007) and dorsal hippocampus (−2.0 through −4.0 from bregma; Paxinos & Watson, 2007) were cut at 30 µm using a freezing microtome. .. WFA staining was performed by washing free-floating sections three times for 5 min in 1×-PBS, the tissue was placed in 3% goat blocking serum (Vector Laboratories) for 1 h and was then incubated overnight at 4°C on a shaker table with fluorescein-conjugated WFA (1:500, Vector Laboratories) in 1×-PBS containing 2% goat serum. .. The tissue was washed three times for 10 min each time in 1×-PBS and mounted onto Frost plus slides in diluted 1×-PBS (30:200) with 0.24% Triton X-100 (Sigma-Aldrich).

    Immunostaining:

    Article Title: Venlafaxine Stimulates an MMP-9-Dependent Increase in Excitatory/Inhibitory Balance in a Stress Model of Depression
    Article Snippet: Sections were washed 2 or 3 times with 1× PBS, then permeabilized with 1× PBS containing 0.1% Triton X-100, blocked with 10% normal goat serum, and incubated with anti-PV (1:500, Sigma Millipore, P3088) overnight at 4°C. .. Following subsequent washes and incubation with a fluorescent secondary antibody for PV immunostaining and fluorescein-labeled Wisteria floribunda lectin (WFA) (1:1000, Vector Laboratories, FL-1351) for 2 h at room temperature, sections were washed several times with 1× PBS, counterstained with DAPI, and mounted with Hydromount (National Diagnostics, HS-106) before being allowed to dry several days at 4°C. .. For PV and PNN cell quantification, images were acquired using a Leica SP8 laser scanning confocal microscope with an oil immersion, 209 objective with 0.40 numerical aperture.

    Binding Assay:

    Article Title: CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation
    Article Snippet: .. Lectin binding assay Fluorescein-labeled GNA, from the snowdrop pea (Vector Laboratories, Burlingame, CA, US), was used to detect the cell surface expression of Man5 glycoforms. ..

    Expressing:

    Article Title: CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation
    Article Snippet: .. Lectin binding assay Fluorescein-labeled GNA, from the snowdrop pea (Vector Laboratories, Burlingame, CA, US), was used to detect the cell surface expression of Man5 glycoforms. ..

    Laser-Scanning Microscopy:

    Article Title: Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms
    Article Snippet: .. After digestion, biofilms were washed, and Pel was then stained with fluorescein-labeled WFL lectin (100 μg/ml; Vector Laboratories) for 15 min. Stained biofilms were washed before visualization on a Zeiss LSM 510 confocal laser scanning microscope. .. Image analysis was conducted using the Velocity software (Improvision).

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  • 93
    Vector Laboratories wfa
    Histologic assessment of neuronal populations in the Ts65Dn visual cortex. A–O , Representative immunostains ( A–L ) and <t>WFA</t> staining ( M–O ) of nontrisomic littermate ( A , D , G , J , M ) and Ts65Dn ( B , E , H , K , N ) visual cortex. No significant differences were observed in the density of NeuN + ( A–C ), Gad67 + ( D–F ), parvalbumin + ( G–I ), <t>somatostatin</t> + ( J–L ), or WFA-stained neurons in Ts65Dn and nontrisomic visual cortex. C , F , I , L , O , Quantitation of cell counts in nontrisomic (black) and Ts65Dn (red) cortex. Scale bar, 500 μm. Error bars denote SD. n = 6 per group.
    Wfa, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wfa/product/Vector Laboratories
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    wfa - by Bioz Stars, 2021-03
    93/100 stars
      Buy from Supplier

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    Histologic assessment of neuronal populations in the Ts65Dn visual cortex. A–O , Representative immunostains ( A–L ) and WFA staining ( M–O ) of nontrisomic littermate ( A , D , G , J , M ) and Ts65Dn ( B , E , H , K , N ) visual cortex. No significant differences were observed in the density of NeuN + ( A–C ), Gad67 + ( D–F ), parvalbumin + ( G–I ), somatostatin + ( J–L ), or WFA-stained neurons in Ts65Dn and nontrisomic visual cortex. C , F , I , L , O , Quantitation of cell counts in nontrisomic (black) and Ts65Dn (red) cortex. Scale bar, 500 μm. Error bars denote SD. n = 6 per group.

    Journal: The Journal of Neuroscience

    Article Title: Activity-Dependent Dysfunction in Visual and Olfactory Sensory Systems in Mouse Models of Down Syndrome

    doi: 10.1523/JNEUROSCI.1045-17.2017

    Figure Lengend Snippet: Histologic assessment of neuronal populations in the Ts65Dn visual cortex. A–O , Representative immunostains ( A–L ) and WFA staining ( M–O ) of nontrisomic littermate ( A , D , G , J , M ) and Ts65Dn ( B , E , H , K , N ) visual cortex. No significant differences were observed in the density of NeuN + ( A–C ), Gad67 + ( D–F ), parvalbumin + ( G–I ), somatostatin + ( J–L ), or WFA-stained neurons in Ts65Dn and nontrisomic visual cortex. C , F , I , L , O , Quantitation of cell counts in nontrisomic (black) and Ts65Dn (red) cortex. Scale bar, 500 μm. Error bars denote SD. n = 6 per group.

    Article Snippet: Sections were stained by overnight incubation with guinea pig anti-Arc (catalog #156 005, Synaptic Systems; 1:1000), mouse anti-NeuN (MAB377, Millipore; 1:1000), mouse anti-parvalbumin (P3088, Sigma-Aldrich; 1:4000), rabbit anti-somatostatin (ab64053, Abcam; 1:1000), and mouse anti-Gad67 (MAB5406, Millipore; 1:1000) antibodies and/or WFA (FL-1351, Vector Laboratories; 1:1000) after 10 min postfixation with 4% PFA, permeabilization with 0.3% Tween 20, and blocking with 10% goat serum.

    Techniques: Staining, Quantitation Assay

    Degradation and reformation of PNNs in the cerebellar nuclei after exposure to ChABC. A: Degradation and reformation of PNN CSPGs in the cerebellar nuclei. Top panels show staining of four different areas of the cerebellar nuclei for CSPGs of perineuronal nets with WFA-Fluorescein. The proposed maximal extent of the spread of the enzyme is indicated in red. The time of the injections before sacrifice of the animal is noted below each column in days. Bottom panels shows the same areas stained with avidin to indicate locations of cerebellar nuclear neurons. Scale bars = 200 µm. Note that the right dentate is at a different scale to accommodate the entire injection. Small regions for which we lacked a photo are filled in with black. Additionally W, X, Y and Z label locations within each image for which we include higher resolution views below. Scale bars = 100 µm B: Estimate of spread of ChABC. Calculated area (in mm 2 ) without WFA-staining from A for each injection site.

    Journal: PLoS ONE

    Article Title: N-Acetylgalactosamine Positive Perineuronal Nets in the Saccade-Related-Part of the Cerebellar Fastigial Nucleus Do Not Maintain Saccade Gain

    doi: 10.1371/journal.pone.0086154

    Figure Lengend Snippet: Degradation and reformation of PNNs in the cerebellar nuclei after exposure to ChABC. A: Degradation and reformation of PNN CSPGs in the cerebellar nuclei. Top panels show staining of four different areas of the cerebellar nuclei for CSPGs of perineuronal nets with WFA-Fluorescein. The proposed maximal extent of the spread of the enzyme is indicated in red. The time of the injections before sacrifice of the animal is noted below each column in days. Bottom panels shows the same areas stained with avidin to indicate locations of cerebellar nuclear neurons. Scale bars = 200 µm. Note that the right dentate is at a different scale to accommodate the entire injection. Small regions for which we lacked a photo are filled in with black. Additionally W, X, Y and Z label locations within each image for which we include higher resolution views below. Scale bars = 100 µm B: Estimate of spread of ChABC. Calculated area (in mm 2 ) without WFA-staining from A for each injection site.

    Article Snippet: We stained another set for intact perineuronal nets with wisteria floribunda agglutinin (WFA) conjugated with fluorescein (1∶500, Vector Labs FL-1351) using standard immunohistochemical techniques .

    Techniques: Staining, Avidin-Biotin Assay, Injection

    The glycoside hydrolases PslG h and PelA h hydrolyze the exopolysaccharides Pel and Psl in a biofilm. Representative confocal images of Psl biofilms grown statically for 24 hours ( top ) and Pel biofilms cultivated for 48 hours ( bottom ) under flow conditions and treated with wild-type hydrolases or hydrolases that have point mutations to catalytic residues. Biofilms were stained with the HHA Psl-specific lectin (green) and WFL Pel-specific lectin (red). Scale bars, 30 μm.

    Journal: Science Advances

    Article Title: Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

    doi: 10.1126/sciadv.1501632

    Figure Lengend Snippet: The glycoside hydrolases PslG h and PelA h hydrolyze the exopolysaccharides Pel and Psl in a biofilm. Representative confocal images of Psl biofilms grown statically for 24 hours ( top ) and Pel biofilms cultivated for 48 hours ( bottom ) under flow conditions and treated with wild-type hydrolases or hydrolases that have point mutations to catalytic residues. Biofilms were stained with the HHA Psl-specific lectin (green) and WFL Pel-specific lectin (red). Scale bars, 30 μm.

    Article Snippet: After digestion, biofilms were washed, and Pel was then stained with fluorescein-labeled WFL lectin (100 μg/ml; Vector Laboratories) for 15 min. Stained biofilms were washed before visualization on a Zeiss LSM 510 confocal laser scanning microscope.

    Techniques: Flow Cytometry, Staining

    GNA probe for cell surface oligomannose glycan expression. GNA binds glycan structures with terminal mannose and will not bind complex, sialic acid–containing glycans. CHO-S cells were transfected with a plasmid designed to inactivate the MGAT1 gene by CRISPR/Cas9 gene editing (MGAT CHO). The cells were treated with fluorescein-conjugated GNA to screen for the incorporation of high-mannose glycans in the cell membrane. HEK 293 GnTI − cells that also lack the MGAT1 gene served as a positive control (panels A and D), while normal CHO-S cells that possess an intact MGAT1 gene served as a negative control (panels B and E). Cells were visualized under 20× magnification on a Leica DM5500 B widefield microscope using DIC (upper panels A, B, C) or under illumination with 495-nm light (lower panels D, E, and F). Cas9, CRISPR-associated protein 9; CHO, Chinese hamster ovary; CRISPR, clustered regularly interspaced short palindromic repeat; DIC, differential interference contrast; GNA, Galanthus nivalis lectin; GnTI, N-acetylglucosaminyltransferase I; HEK 293, human embryonic kidney 293; MGAT1, Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase.

    Journal: PLoS Biology

    Article Title: CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation

    doi: 10.1371/journal.pbio.2005817

    Figure Lengend Snippet: GNA probe for cell surface oligomannose glycan expression. GNA binds glycan structures with terminal mannose and will not bind complex, sialic acid–containing glycans. CHO-S cells were transfected with a plasmid designed to inactivate the MGAT1 gene by CRISPR/Cas9 gene editing (MGAT CHO). The cells were treated with fluorescein-conjugated GNA to screen for the incorporation of high-mannose glycans in the cell membrane. HEK 293 GnTI − cells that also lack the MGAT1 gene served as a positive control (panels A and D), while normal CHO-S cells that possess an intact MGAT1 gene served as a negative control (panels B and E). Cells were visualized under 20× magnification on a Leica DM5500 B widefield microscope using DIC (upper panels A, B, C) or under illumination with 495-nm light (lower panels D, E, and F). Cas9, CRISPR-associated protein 9; CHO, Chinese hamster ovary; CRISPR, clustered regularly interspaced short palindromic repeat; DIC, differential interference contrast; GNA, Galanthus nivalis lectin; GnTI, N-acetylglucosaminyltransferase I; HEK 293, human embryonic kidney 293; MGAT1, Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase.

    Article Snippet: Lectin binding assay Fluorescein-labeled GNA, from the snowdrop pea (Vector Laboratories, Burlingame, CA, US), was used to detect the cell surface expression of Man5 glycoforms.

    Techniques: Expressing, Transfection, Plasmid Preparation, CRISPR, Positive Control, Negative Control, Microscopy

    Flow chart of MGAT1 gene editing and cell line selection strategy. (A) A plasmid containing the Cas9 nuclease, tracrRNA, and a gRNA sequence was electroporated into suspension adapted CHO-S cells. (B) Twenty-four hours following transfection, the cells were distributed into 96-well tissue culture plates at a density of 0.5 cells/well. (C) Between 12 and 15 days later, wells with 20% or greater confluency were transferred to 24-well plates. (D) After 5 days of growth in 24-well plates, a 0.2-mL aliquot was removed from each well, and cells were tested for the ability to bind fluorescein-labeled GNA. (E) GNA-binding cells were then expanded to shake flasks, and cell lines were transiently transfected with a gene encoding A244-rgp120. The cell culture supernatants were then collected after 5 days and tested for binding of gp120 to the prototypic glycan-dependent, broadly neutralizing monoclonal antibody PG9. This representative plot (F) is shown for demonstrative process purposes only. A detailed plot of this data is show in Fig 4A . (G) The gene encoding MGAT1 was sequenced from GNA-binding cell lines that exhibited robust growth and the ability to secrete PG9-binding gp120. The specific mutations induced by NHEJR were determined by Sanger sequencing. Cas9, CRISPR-associated protein 9; CHO, Chinese hamster ovary; FIA, fluorescence immunoassay; GNA, Galanthus nivalis lectin; gRNA, guide RNA; MGAT1, Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase; NHEJR, nonhomologous end joining repair; rgp120, recombinant gp120; tracrRNA, trans -activating CRISPR RNA.

    Journal: PLoS Biology

    Article Title: CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation

    doi: 10.1371/journal.pbio.2005817

    Figure Lengend Snippet: Flow chart of MGAT1 gene editing and cell line selection strategy. (A) A plasmid containing the Cas9 nuclease, tracrRNA, and a gRNA sequence was electroporated into suspension adapted CHO-S cells. (B) Twenty-four hours following transfection, the cells were distributed into 96-well tissue culture plates at a density of 0.5 cells/well. (C) Between 12 and 15 days later, wells with 20% or greater confluency were transferred to 24-well plates. (D) After 5 days of growth in 24-well plates, a 0.2-mL aliquot was removed from each well, and cells were tested for the ability to bind fluorescein-labeled GNA. (E) GNA-binding cells were then expanded to shake flasks, and cell lines were transiently transfected with a gene encoding A244-rgp120. The cell culture supernatants were then collected after 5 days and tested for binding of gp120 to the prototypic glycan-dependent, broadly neutralizing monoclonal antibody PG9. This representative plot (F) is shown for demonstrative process purposes only. A detailed plot of this data is show in Fig 4A . (G) The gene encoding MGAT1 was sequenced from GNA-binding cell lines that exhibited robust growth and the ability to secrete PG9-binding gp120. The specific mutations induced by NHEJR were determined by Sanger sequencing. Cas9, CRISPR-associated protein 9; CHO, Chinese hamster ovary; FIA, fluorescence immunoassay; GNA, Galanthus nivalis lectin; gRNA, guide RNA; MGAT1, Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase; NHEJR, nonhomologous end joining repair; rgp120, recombinant gp120; tracrRNA, trans -activating CRISPR RNA.

    Article Snippet: Lectin binding assay Fluorescein-labeled GNA, from the snowdrop pea (Vector Laboratories, Burlingame, CA, US), was used to detect the cell surface expression of Man5 glycoforms.

    Techniques: Flow Cytometry, Selection, Plasmid Preparation, Sequencing, Transfection, Labeling, Binding Assay, Cell Culture, CRISPR, Fluorescence, Recombinant