Gtx635679

Manufactured by GeneTex

GTX635679 is a laboratory instrument designed for DNA sequencing. It utilizes advanced optical detection technology to accurately read and analyze DNA samples. The core function of this equipment is to facilitate high-throughput, precise genetic analysis without further interpretation.

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Lab products found in correlation

Alexa fluor 488 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (5 mentions) Cytation 5, by Agilent Technologies (5 mentions) 96 well plate, by Corning (4 mentions) Gtx632604, by GeneTex (2 mentions) Affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions) Ab9535, by Abcam (1 mentions) Ab134131, by Abcam (1 mentions) Ab255822, by Abcam (1 mentions) Ab125212, by Abcam (1 mentions) Dab substrate chromogen system, by Agilent Technologies (1 mentions) Goat anti mouse immunoglobulins hrp, by Agilent Technologies (1 mentions) Ab28364, by Abcam (1 mentions) Goat anti rabbit immunoglobulins hrp, by Agilent Technologies (1 mentions) Rabbit anti rat immunoglobulins hrp, by Agilent Technologies (1 mentions) Alexa fluor 488 conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions) Ix73 fluorescence microscope, by Olympus (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) A21428, by Thermo Fisher Scientific (1 mentions) Cellinsight cx7 hcs, by Thermo Fisher Scientific (1 mentions) A11001, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

HL344] (GTX635679) (2 citations)

11 protocols using gtx635679

Histopathological Analysis of SARS-CoV-2 Infection

Cited 2 times

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The lung (right upper lobe), brain (right hemisphere) and nasal turbinates were collected and placed in 10% formalin for >72 hrs. Fixed tissues were embedded in paraffin and tissue sections (3 μm) were processed for histology and immunohistochemistry (IHC). For histologic examination, slides were stained with hematoxylin & eosin (H&E). Scoring of lung histopathological features was based on¹⁸ (link)^,¹⁹ (link) and detailed in Table 3. Quantitative morphometric analysis of lung pathology (% Lesion) was performed using two softwares: Interactive Learning and Segmentation Toolkit (Ilastik version 1.3.3) and CellProfiler Analyst software (version 3.1.5). Probability maps for the lesion area and whole lung are created in Ilastik and analysed in the CellProfiler Analyst software.⁷¹ (link) For viral detection, an IHC assay was performed using an anti-SARS-CoV-2 nucleocapsid protein rabbit IgG monoclonal antibody (GeneTex; GTX635679) at 1:1000 dilution and a peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, 111-035-144) at 1:500 dilution. Images were acquired with a NanoZoomer 2.0- HT Whole Slide Imager, Digital Pathology Slide Scanner.

Gonçalves R., Couto J., Ferreirinha P., Costa J.M., Silvério D., Silva M.L., Fernandes A.I., Madureira P., Alves N.L., Lamas S, & Saraiva M. (2023). SARS-CoV-2 variants induce distinct disease and impact in the bone marrow and thymus of mice. iScience, 26(2), 105972.

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SARS-CoV-2 Infection Assay in Huh7-hACE2 Cells

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Huh7-hACE2 cells in 96-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% fetal bovine serum (FBS). Before 1.5 hr viral inoculation, the tested compounds were added to the wells in triplicate. The infection proceeded for 24 hr without the removal of the viruses or the compounds. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific). Hoechst 33342 was added in the final step to counterstain the nuclei. Fluorescence images of approximately 10,000 cells were acquired per well with a 10× objective in a Cytation 5 (BioTek). The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek).

Seifert M., Bera S.C., van Nies P., Kirchdoerfer R.N., Shannon A., Le T.T., Meng X., Xia H., Wood J.M., Harris L.D., Papini F.S., Arnold J.J., Almo S., Grove T.L., Shi P.Y., Xiang Y., Canard B., Depken M., Cameron C.E, & Dulin D. (2021). Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective. eLife, 10, e70968.

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SARS-CoV-2 Antiviral Immunofluorescence Assay

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An antiviral
immunofluorescence
assay was carried out as previously described.^{8 (link),36 (link)} Briefly,
Vero E6 cells or Caca2-hACE2 cells in 96-well plates (Corning) were
infected with SARS-CoV-2 (USA-WA1/2020 isolate) at a MOI of 0.1 in
DMEM supplemented with 1% FBS. Immediately before the viral inoculation,
the tested compounds in a 3-fold dilution concentration series were
also added to the wells in triplicate. The infection proceeded for
24 h without the removal of the viruses or the compounds. The staining
and quantification procedures are described in our previous publications.^{8 (link)} Briefly, the cells were fixed with 4% paraformaldehyde,
permeabilized with 0.1% Triton-100, blocked with DMEM containing 10%
FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2
NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat antimouse
secondary antibody (ThermoFisher Scientific). Hoechst 33342 was added
in the final step to counterstain the nuclei. Fluorescence images
of approximately 10 000 cells were acquired per well with a
10× objective in a Cytation 5 (BioTek). The total number of cells,
as indicated by the nuclei staining, and the fraction of the infected
cells, as indicated by the NP staining, were quantified with the cellular
analysis module of the Gen5 software (BioTek).

Ma C., Sacco M.D., Xia Z., Lambrinidis G., Townsend J.A., Hu Y., Meng X., Szeto T., Ba M., Zhang X., Gongora M., Zhang F., Marty M.T., Xiang Y., Kolocouris A., Chen Y, & Wang J. (2021). Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay. ACS Central Science, 7(7), 1245-1260.

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SARS-CoV-2 Immunohistochemistry in Lung Tissue

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The formalin‐fixed paraffin‐embedded lung tissue sections (4.25 μm in thickness) were stained with hematoxylin and eosin (H&E). An immunohistochemistry assay was employed using several primary antibodies against SARS‐CoV‐2 spike protein (mouse, GTX632604, GeneTex), SARS‐CoV‐2 nucleocapsid protein (rabbit, GTX635679, GeneTex), CD31 (rabbit, ab28364, Abcam), CD41 (rabbit, ab134131, Abcam), CD45 (rat, #103202, Biolegend), MPO (rabbit, ab9535, Abcam), CD68 (rabbit, ab125212, Abcam), and P‐selectin/CD62P (rabbit, ab255822, Abcam). The used secondary antibodies include Goat Anti‐Rabbit Immunoglobulins/HRP (P0448, Dako), Goat Anti‐Mouse Immunoglobulins/HRP (P0447, Dako), and Rabbit Anti‐Rat Immunoglobulins/HRP (P0450, Dako). The liquid DAB⁺ Substrate Chromogen System (K3468, Dako) was used for color development. DAB signal‐positive areas of CD41, CD45, MPO, and CD68 were quantified using an ImageJ software (NIH). The area with a particularly high (N) protein positive signal was defined as “N protein signal‐rich area,” and the positive areas of CD41, CD45, MPO, and CD68 were quantified using an ImageJ software (NIH).

Tsumita T., Takeda R., Maishi N., Hida Y., Sasaki M., Orba Y., Sato A., Toba S., Ito W., Teshirogi T., Sakurai Y., Iba T., Naito H., Ando H., Watanabe H., Mizuno A., Nakanishi T., Matsuda A., Zixiao R., Lee J., Iimura T., Sawa H, & Hida K. (2023). Viral uptake and pathophysiology of the lung endothelial cells in age‐associated severe SARS‐CoV‐2 infection models. Aging Cell, 23(2), e14050.

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SARS-CoV-2 Antiviral Immunofluorescence Assay

Cited 1 time

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Antiviral immunofluorescence assay was carried out as previously described.^{8 (link)} Briefly, Vero E6 cells in 96-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in DMEM supplemented with 1% FBS. Immediately before the viral inoculation, the tested compounds in a three-fold dilution concentration series were also added to the wells in triplicate. The infection proceeded for 48 h without the removal of the viruses or the compounds. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific). Hoechst 33342 was added in the final step to counterstain the nuclei. Fluorescence images of approximately ten thousand cells were acquired per well with a 10x objective in a Cytation 5 (BioTek). The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek).

Kitamura N., Sacco M.D., Ma C., Hu Y., Townsend J.A., Meng X., Zhang F., Zhang X., Ba M., Szeto T., Kukuljac A., Marty M.T., Schultz D., Cherry S., Xiang Y., Chen Y, & Wang J. (2021). Expedited approach toward the rational design of non-covalent SARS-CoV-2 main protease inhibitors. Journal of medicinal chemistry, 65(4), 2848-2865.

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SARS-CoV-2 Infection Assay in Vero E6 Cells

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Vero E6 cells in 96-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at a multiplicity of infection (MOI) of 0.05 in DMEM supplemented with 1% FBS. Immediately before the viral inoculation, the tested compounds in a threefold dilution concentration series were also added to the wells in triplicate. The infection proceeded for 48 hours without the removal of the viruses or the compounds. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 nucleoprotein (NP) (GeneTex, GTX635679) and an Alexa Fluor 488–conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific). Hoechst 33342 was added in the final step to counterstain the nuclei. Fluorescence images of approximately 10,000 cells were acquired per well with a 10× objective in a Cytation 5 (BioTek). The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek).

Sacco M.D., Ma C., Lagarias P., Gao A., Townsend J.A., Meng X., Dube P., Zhang X., Hu Y., Kitamura N., Hurst B., Tarbet B., Marty M.T., Kolocouris A., Xiang Y., Chen Y, & Wang J. (2020). Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against Mpro and cathepsin L. Science Advances, 6(50), eabe0751.

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SARS-CoV-2 Infection Assay in Vero-TMPRSS2 and Calu-3 Cells

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Vero-TMPRSS2 and Calu-3 cells were inoculated with SARS-CoV-2 at MOIs of 0.1 and 10, respectively. After a 1 hour incubation, cells were fed with fresh culture medium containing 2% FBS and S-217622. Vero-TMPRSS2 cells at 24 hpi and Calu-3 cells at 72 hpi were fixed with 3.7% formaldehyde in PBS. The MOIs and harvest time points were optimized to achieve similar infection rates in different cell lines. Cells were then permeabilized with 0.5% Triton X-100 in PBS for 5 minutes and stained with anti-SARS-CoV-2 nucleocapsid rabbit monoclonal antibody (1:1000, GTX635679, GeneTex) in 25% Block Ace (KAC) in PBS for 1 hour. Alexa Fluor 488-conjugated anti-rabbit IgG antibody (1:1000, Invitrogen; Thermo Fisher Scientific) was used as the secondary antibody. Nuclei were stained with Hoechst 33342 (Invitrogen). Fluorescent images were captured using IX73 fluorescence microscope (Olympus).

Sasaki M., Tabata K., Kishimoto M., Itakura Y., Kobayashi H., Ariizumi T., Uemura K., Toba S., Kusakabe S., Maruyama Y., Iida S., Nakajima N., Suzuki T., Yoshida S., Nobori H., Sanaki T., Kato T., Shishido T., Hall W.W., Orba Y., Sato A, & Sawa H. (2022). S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and ameliorates COVID-19 severity in hamsters. Science Translational Medicine.

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SARS-CoV-2 Infection Assay in Vero E6 Cells

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Vero E6 cells in 96-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in DMEM supplemented with 1% FBS. Immediately before the viral inoculation, the tested compounds in a three-fold dilution concentration series were also added to the wells in triplicate. The infection proceeded for 48 h without the removal of the viruses or the compounds. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific). Hoechst 33342 was added in the final step to counterstain the nuclei. Fluorescence images of approximately ten thousand cells were acquired per well with a 10x objective in a Cytation 5 (BioTek). The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek).

Sacco M.D., Ma C., Lagarias P., Gao A., Townsend J.A., Meng X., Dube P., Zhang X., Hu Y., Kitamura N., Hurst B., Tarbet B., Marty M.T., Kolocouris A., Xiang Y., Chen Y, & Wang J. (2020). Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L. bioRxiv.

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Quantification of SARS-CoV-2 Omicron Infection

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SARS-CoV-2 Omicron BA.2 or BA.5 stocks were produced as described in supplementary materials M7 and diluted in cell-specific media to a multiplicity of infection (MOI) of 2. Caco-2 cells were seeded at a concentration of 8,000 cells/well in 96-well plates the day before infection. Cells were pre-treated with compounds and then incubated with the virus, followed by fixation of the cells with 4% formalin in PBS for 30 min. Cells were subsequently washed with PBS, permeabilized with 0.1% Triton X-100 for 10 min and blocked with 1% BSA in PBS for 1 h at RT, followed by immunostaining with the mouse primary dsRNA antibody (J2-1904, Scicons English and Scientific Consulting) and rabbit primary SARS-CoV-2 antibody (HL344, Genetex GTX635679) at working dilutions of 1:1000 in 1% BSA in PBS overnight at 4°C. Secondary antibodies were used at a 1:2000 dilution in 1% BSA in PBS and included the goat anti-mouse IgG Alexa Fluor 488 (A11001, Invitrogen) and goat anti-rabbit IgG Alexa Fluor 555 (A21428, Invitrogen) with the nuclear stain Hoechst 33342 at 1.5 µg/mL for 1 h at RT. After washing with PBS, the plates were imaged on a high-content screening (HCS) platform (CellInsight CX7 HCS, Thermo Fisher Scientific) with a 10X objective.

Cloherty A.P., Rader A.G., Patel K.S., Pérez-Vargas J., Thompson C.A., Ennis S., Niikura M., Wildenberg M.E., Muncan V., Schreurs R.R., Jean F, & Ribeiro C.M. (2023). Berbamine suppresses intestinal SARS-CoV-2 infection via a BNIP3-dependent autophagy blockade. Emerging Microbes & Infections, 12(1), 2195020.

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Histopathological Analysis of SARS-CoV-2 Lung Tissue

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Lung tissue was collected, fixed in 10% neutral buffered formalin, and submitted to the Cornell University Animal Health Diagnostic Center for sectioning and staining. Tissue sections (4 μm) were stained with H&E and examined by a board-certified veterinary anatomic pathologist blinded to group categories. Lung pathology was scored based on the percentage of each feature affected across the entire lung section. The features scored were perivascular infiltrates, interstitial infiltrates, presence of cells in alveoli lumina, and type II pneumocyte hyperplasia, the presence of sloughed or necrotic cells in bronchiolar lumina. Each point corresponded to the amount of tissue affected: normal (0); less than 10% (1 ); between 10 and 25% (2 ); 26 to 50% (3 (link)); and higher than 50% (4 (link)). As described previously, IHC was performed on lung tissue with an anti–SARS-CoV-2 nucleoprotein antibody (GeneTex: GTX635679) at a 1:5000 dilution (58 (link)).

Imbiakha B., Sahler J.M., Buchholz D.W., Ezzatpour S., Jager M., Choi A., Monreal I.A., Byun H., Adeleke R.A., Leach J., Whittaker G., Dewhurst S., Rudd B.D., Aguilar H.C, & August A. (2024). Adaptive immune cells are necessary for SARS-CoV-2–induced pathology. Science Advances, 10(1), eadg5461.

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