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hypercyt tm high throughput flow cytometry platform  (Intellicyt)

 
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    Intellicyt hypercyt tm high throughput flow cytometry platform
    Flow <t>cytometry</t> assay for the HEG1–KRIT1 FERM domain interaction. (A) Ribbon diagram of KRIT1 FERM domain in complex with the HEG1 cytoplasmic tail (PDB ID: 3u7d). The HEG1 peptide is shown in yellow. The KRIT1 FERM domain consists of three subdomains: F1 (green and blue); F2 (red); and F3 (orange). The feature of the F1 domain that is not present in other FERM domain is shown in blue and that region is an important part of the HEG1 binding pocket. (B) Schematic representation of the HEG1 cytoplasmic tail (a.a. 1274–1381) peptide coupled to Neutravidin beads and the EGFP‐KRIT1 FERM domain. Binding of EGFP‐KRIT1 FERM domain to the HEG1 matrix beads can be detected by flow cytometry. Small‐molecule inhibitors HKi preventing the interaction of EGFP‐KRIT1 FERM domain with the HEG1 matrix beads reduce the EGFP fluorescence signal. (C) Flow cytometry profile of SPHERO Neutravidin Polystyrene Particles coated with increasing amount of biotinylated HEG1 peptide and 150 nM EGFP‐KRIT1 FERM domain. We noticed many beads doublets in the light scatter signal at 1,500 nM concentration of HEG1 peptide. (D) Titration curve for the interaction of EGFP‐KRIT1 FERM domain with increasing amounts of HEG1 on the beads as shown in panel C, as measured by geometric mean fluorescence intensity (GMFI). We used the 150 nM HEG1 peptide concentration for future experiments. (E) Titration curve for the interaction of 150 nM HEG1 on the beads with increasing amounts of EGFP‐KRIT1 FERM domain (0–250 nM) wild‐type (blue line) and KRIT1(L717,721A) mutant (red line). We used the 70 nM EGFP‐KRIT1 concentration for future experiments. (F) Competition binding curve of 70 nM EGFP‐KRIT1 FERM domain binding to 150 nM HEG1 on the beads with increasing amounts on non‐biotinylated HEG1 7‐mer peptide. We used the 2 μM HEG1 7‐mer concentration for future experiments.
    Hypercyt Tm High Throughput Flow Cytometry Platform, supplied by Intellicyt, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hypercyt tm high throughput flow cytometry platform/product/Intellicyt
    Average 90 stars, based on 1 article reviews
    hypercyt tm high throughput flow cytometry platform - by Bioz Stars, 2026-06
    90/100 stars

    Images

    1) Product Images from "Inhibition of the HEG1–KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells"

    Article Title: Inhibition of the HEG1–KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells

    Journal: FASEB BioAdvances

    doi: 10.1096/fba.2020-00141

    Flow cytometry assay for the HEG1–KRIT1 FERM domain interaction. (A) Ribbon diagram of KRIT1 FERM domain in complex with the HEG1 cytoplasmic tail (PDB ID: 3u7d). The HEG1 peptide is shown in yellow. The KRIT1 FERM domain consists of three subdomains: F1 (green and blue); F2 (red); and F3 (orange). The feature of the F1 domain that is not present in other FERM domain is shown in blue and that region is an important part of the HEG1 binding pocket. (B) Schematic representation of the HEG1 cytoplasmic tail (a.a. 1274–1381) peptide coupled to Neutravidin beads and the EGFP‐KRIT1 FERM domain. Binding of EGFP‐KRIT1 FERM domain to the HEG1 matrix beads can be detected by flow cytometry. Small‐molecule inhibitors HKi preventing the interaction of EGFP‐KRIT1 FERM domain with the HEG1 matrix beads reduce the EGFP fluorescence signal. (C) Flow cytometry profile of SPHERO Neutravidin Polystyrene Particles coated with increasing amount of biotinylated HEG1 peptide and 150 nM EGFP‐KRIT1 FERM domain. We noticed many beads doublets in the light scatter signal at 1,500 nM concentration of HEG1 peptide. (D) Titration curve for the interaction of EGFP‐KRIT1 FERM domain with increasing amounts of HEG1 on the beads as shown in panel C, as measured by geometric mean fluorescence intensity (GMFI). We used the 150 nM HEG1 peptide concentration for future experiments. (E) Titration curve for the interaction of 150 nM HEG1 on the beads with increasing amounts of EGFP‐KRIT1 FERM domain (0–250 nM) wild‐type (blue line) and KRIT1(L717,721A) mutant (red line). We used the 70 nM EGFP‐KRIT1 concentration for future experiments. (F) Competition binding curve of 70 nM EGFP‐KRIT1 FERM domain binding to 150 nM HEG1 on the beads with increasing amounts on non‐biotinylated HEG1 7‐mer peptide. We used the 2 μM HEG1 7‐mer concentration for future experiments.
    Figure Legend Snippet: Flow cytometry assay for the HEG1–KRIT1 FERM domain interaction. (A) Ribbon diagram of KRIT1 FERM domain in complex with the HEG1 cytoplasmic tail (PDB ID: 3u7d). The HEG1 peptide is shown in yellow. The KRIT1 FERM domain consists of three subdomains: F1 (green and blue); F2 (red); and F3 (orange). The feature of the F1 domain that is not present in other FERM domain is shown in blue and that region is an important part of the HEG1 binding pocket. (B) Schematic representation of the HEG1 cytoplasmic tail (a.a. 1274–1381) peptide coupled to Neutravidin beads and the EGFP‐KRIT1 FERM domain. Binding of EGFP‐KRIT1 FERM domain to the HEG1 matrix beads can be detected by flow cytometry. Small‐molecule inhibitors HKi preventing the interaction of EGFP‐KRIT1 FERM domain with the HEG1 matrix beads reduce the EGFP fluorescence signal. (C) Flow cytometry profile of SPHERO Neutravidin Polystyrene Particles coated with increasing amount of biotinylated HEG1 peptide and 150 nM EGFP‐KRIT1 FERM domain. We noticed many beads doublets in the light scatter signal at 1,500 nM concentration of HEG1 peptide. (D) Titration curve for the interaction of EGFP‐KRIT1 FERM domain with increasing amounts of HEG1 on the beads as shown in panel C, as measured by geometric mean fluorescence intensity (GMFI). We used the 150 nM HEG1 peptide concentration for future experiments. (E) Titration curve for the interaction of 150 nM HEG1 on the beads with increasing amounts of EGFP‐KRIT1 FERM domain (0–250 nM) wild‐type (blue line) and KRIT1(L717,721A) mutant (red line). We used the 70 nM EGFP‐KRIT1 concentration for future experiments. (F) Competition binding curve of 70 nM EGFP‐KRIT1 FERM domain binding to 150 nM HEG1 on the beads with increasing amounts on non‐biotinylated HEG1 7‐mer peptide. We used the 2 μM HEG1 7‐mer concentration for future experiments.

    Techniques Used: Flow Cytometry, Binding Assay, Fluorescence, Concentration Assay, Titration, Mutagenesis

    HKi2 treatment leads to KLF2 and KLF4 upregulation in endothelial cells. (A‐F) hCMEC/D3 cells treated with HKi2 (50 μM) or vehicle control and analyzed by qPCR for mRNA level. (A, B) Dose response of KLF4 and KLF2 mRNA expression at indicated doses for 12 h. HKi2 induces KLF4 and KLF2 mRNA expression at indicated concentrations. (C, D) Timecourse, HKi2 induces a rapid and sustained upregulation of KLF4 and KLF2 mRNA expression. (E,F) HKi2 treatment for 4 h upregulated (E) KLF4, and (F) KLF2, and an inactive compound, 2‐hydroxy‐1‐naphthoic acid (50 μM) (compound 9), did not. (A‐F) Bar graphs represent mRNA levels relative to vehicle control ± SEM with: (A‐D) n = 3, t test and (E,F) n = 4, one‐way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (G) Representative images of hCMEC/D3 cells treated for 12 h with HKi2 (50 μM) or inactive compound (50 μM), 2‐hydroxy‐1‐naphthoic acid, and analyzed by Immunofluorescence for KLF4 protein levels. KLF4 expression is increased after treatment with HKi2. (H) Cell viability as assessed by flow cytometry using Propidium Iodide staining, no significant difference between vehicle, HKi2 and inactive compound, 2‐hydroxy‐1‐naphthoic acid. Percentage of viable cells ± SEM. n=3, one‐way ANOVA.
    Figure Legend Snippet: HKi2 treatment leads to KLF2 and KLF4 upregulation in endothelial cells. (A‐F) hCMEC/D3 cells treated with HKi2 (50 μM) or vehicle control and analyzed by qPCR for mRNA level. (A, B) Dose response of KLF4 and KLF2 mRNA expression at indicated doses for 12 h. HKi2 induces KLF4 and KLF2 mRNA expression at indicated concentrations. (C, D) Timecourse, HKi2 induces a rapid and sustained upregulation of KLF4 and KLF2 mRNA expression. (E,F) HKi2 treatment for 4 h upregulated (E) KLF4, and (F) KLF2, and an inactive compound, 2‐hydroxy‐1‐naphthoic acid (50 μM) (compound 9), did not. (A‐F) Bar graphs represent mRNA levels relative to vehicle control ± SEM with: (A‐D) n = 3, t test and (E,F) n = 4, one‐way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (G) Representative images of hCMEC/D3 cells treated for 12 h with HKi2 (50 μM) or inactive compound (50 μM), 2‐hydroxy‐1‐naphthoic acid, and analyzed by Immunofluorescence for KLF4 protein levels. KLF4 expression is increased after treatment with HKi2. (H) Cell viability as assessed by flow cytometry using Propidium Iodide staining, no significant difference between vehicle, HKi2 and inactive compound, 2‐hydroxy‐1‐naphthoic acid. Percentage of viable cells ± SEM. n=3, one‐way ANOVA.

    Techniques Used: Control, Expressing, Immunofluorescence, Flow Cytometry, Staining

    The aldehyde in position C1 and hydroxyl group in position C2 are important for HKi2 activity. The IC 50 was measured using a flow cytometry‐screening assay. >500 = no inhibition detected up to 500 μM concentration thus IC 50 >500 μM.
    Figure Legend Snippet: The aldehyde in position C1 and hydroxyl group in position C2 are important for HKi2 activity. The IC 50 was measured using a flow cytometry‐screening assay. >500 = no inhibition detected up to 500 μM concentration thus IC 50 >500 μM.

    Techniques Used: Activity Assay, Flow Cytometry, Screening Assay, Inhibition, Concentration Assay



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    Image Search Results


    Flow cytometry assay for the HEG1–KRIT1 FERM domain interaction. (A) Ribbon diagram of KRIT1 FERM domain in complex with the HEG1 cytoplasmic tail (PDB ID: 3u7d). The HEG1 peptide is shown in yellow. The KRIT1 FERM domain consists of three subdomains: F1 (green and blue); F2 (red); and F3 (orange). The feature of the F1 domain that is not present in other FERM domain is shown in blue and that region is an important part of the HEG1 binding pocket. (B) Schematic representation of the HEG1 cytoplasmic tail (a.a. 1274–1381) peptide coupled to Neutravidin beads and the EGFP‐KRIT1 FERM domain. Binding of EGFP‐KRIT1 FERM domain to the HEG1 matrix beads can be detected by flow cytometry. Small‐molecule inhibitors HKi preventing the interaction of EGFP‐KRIT1 FERM domain with the HEG1 matrix beads reduce the EGFP fluorescence signal. (C) Flow cytometry profile of SPHERO Neutravidin Polystyrene Particles coated with increasing amount of biotinylated HEG1 peptide and 150 nM EGFP‐KRIT1 FERM domain. We noticed many beads doublets in the light scatter signal at 1,500 nM concentration of HEG1 peptide. (D) Titration curve for the interaction of EGFP‐KRIT1 FERM domain with increasing amounts of HEG1 on the beads as shown in panel C, as measured by geometric mean fluorescence intensity (GMFI). We used the 150 nM HEG1 peptide concentration for future experiments. (E) Titration curve for the interaction of 150 nM HEG1 on the beads with increasing amounts of EGFP‐KRIT1 FERM domain (0–250 nM) wild‐type (blue line) and KRIT1(L717,721A) mutant (red line). We used the 70 nM EGFP‐KRIT1 concentration for future experiments. (F) Competition binding curve of 70 nM EGFP‐KRIT1 FERM domain binding to 150 nM HEG1 on the beads with increasing amounts on non‐biotinylated HEG1 7‐mer peptide. We used the 2 μM HEG1 7‐mer concentration for future experiments.

    Journal: FASEB BioAdvances

    Article Title: Inhibition of the HEG1–KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells

    doi: 10.1096/fba.2020-00141

    Figure Lengend Snippet: Flow cytometry assay for the HEG1–KRIT1 FERM domain interaction. (A) Ribbon diagram of KRIT1 FERM domain in complex with the HEG1 cytoplasmic tail (PDB ID: 3u7d). The HEG1 peptide is shown in yellow. The KRIT1 FERM domain consists of three subdomains: F1 (green and blue); F2 (red); and F3 (orange). The feature of the F1 domain that is not present in other FERM domain is shown in blue and that region is an important part of the HEG1 binding pocket. (B) Schematic representation of the HEG1 cytoplasmic tail (a.a. 1274–1381) peptide coupled to Neutravidin beads and the EGFP‐KRIT1 FERM domain. Binding of EGFP‐KRIT1 FERM domain to the HEG1 matrix beads can be detected by flow cytometry. Small‐molecule inhibitors HKi preventing the interaction of EGFP‐KRIT1 FERM domain with the HEG1 matrix beads reduce the EGFP fluorescence signal. (C) Flow cytometry profile of SPHERO Neutravidin Polystyrene Particles coated with increasing amount of biotinylated HEG1 peptide and 150 nM EGFP‐KRIT1 FERM domain. We noticed many beads doublets in the light scatter signal at 1,500 nM concentration of HEG1 peptide. (D) Titration curve for the interaction of EGFP‐KRIT1 FERM domain with increasing amounts of HEG1 on the beads as shown in panel C, as measured by geometric mean fluorescence intensity (GMFI). We used the 150 nM HEG1 peptide concentration for future experiments. (E) Titration curve for the interaction of 150 nM HEG1 on the beads with increasing amounts of EGFP‐KRIT1 FERM domain (0–250 nM) wild‐type (blue line) and KRIT1(L717,721A) mutant (red line). We used the 70 nM EGFP‐KRIT1 concentration for future experiments. (F) Competition binding curve of 70 nM EGFP‐KRIT1 FERM domain binding to 150 nM HEG1 on the beads with increasing amounts on non‐biotinylated HEG1 7‐mer peptide. We used the 2 μM HEG1 7‐mer concentration for future experiments.

    Article Snippet: Assay plates were sampled using the HyperCyt TM high throughput flow cytometry platform (Intellicyt).

    Techniques: Flow Cytometry, Binding Assay, Fluorescence, Concentration Assay, Titration, Mutagenesis

    HKi2 treatment leads to KLF2 and KLF4 upregulation in endothelial cells. (A‐F) hCMEC/D3 cells treated with HKi2 (50 μM) or vehicle control and analyzed by qPCR for mRNA level. (A, B) Dose response of KLF4 and KLF2 mRNA expression at indicated doses for 12 h. HKi2 induces KLF4 and KLF2 mRNA expression at indicated concentrations. (C, D) Timecourse, HKi2 induces a rapid and sustained upregulation of KLF4 and KLF2 mRNA expression. (E,F) HKi2 treatment for 4 h upregulated (E) KLF4, and (F) KLF2, and an inactive compound, 2‐hydroxy‐1‐naphthoic acid (50 μM) (compound 9), did not. (A‐F) Bar graphs represent mRNA levels relative to vehicle control ± SEM with: (A‐D) n = 3, t test and (E,F) n = 4, one‐way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (G) Representative images of hCMEC/D3 cells treated for 12 h with HKi2 (50 μM) or inactive compound (50 μM), 2‐hydroxy‐1‐naphthoic acid, and analyzed by Immunofluorescence for KLF4 protein levels. KLF4 expression is increased after treatment with HKi2. (H) Cell viability as assessed by flow cytometry using Propidium Iodide staining, no significant difference between vehicle, HKi2 and inactive compound, 2‐hydroxy‐1‐naphthoic acid. Percentage of viable cells ± SEM. n=3, one‐way ANOVA.

    Journal: FASEB BioAdvances

    Article Title: Inhibition of the HEG1–KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells

    doi: 10.1096/fba.2020-00141

    Figure Lengend Snippet: HKi2 treatment leads to KLF2 and KLF4 upregulation in endothelial cells. (A‐F) hCMEC/D3 cells treated with HKi2 (50 μM) or vehicle control and analyzed by qPCR for mRNA level. (A, B) Dose response of KLF4 and KLF2 mRNA expression at indicated doses for 12 h. HKi2 induces KLF4 and KLF2 mRNA expression at indicated concentrations. (C, D) Timecourse, HKi2 induces a rapid and sustained upregulation of KLF4 and KLF2 mRNA expression. (E,F) HKi2 treatment for 4 h upregulated (E) KLF4, and (F) KLF2, and an inactive compound, 2‐hydroxy‐1‐naphthoic acid (50 μM) (compound 9), did not. (A‐F) Bar graphs represent mRNA levels relative to vehicle control ± SEM with: (A‐D) n = 3, t test and (E,F) n = 4, one‐way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (G) Representative images of hCMEC/D3 cells treated for 12 h with HKi2 (50 μM) or inactive compound (50 μM), 2‐hydroxy‐1‐naphthoic acid, and analyzed by Immunofluorescence for KLF4 protein levels. KLF4 expression is increased after treatment with HKi2. (H) Cell viability as assessed by flow cytometry using Propidium Iodide staining, no significant difference between vehicle, HKi2 and inactive compound, 2‐hydroxy‐1‐naphthoic acid. Percentage of viable cells ± SEM. n=3, one‐way ANOVA.

    Article Snippet: Assay plates were sampled using the HyperCyt TM high throughput flow cytometry platform (Intellicyt).

    Techniques: Control, Expressing, Immunofluorescence, Flow Cytometry, Staining

    The aldehyde in position C1 and hydroxyl group in position C2 are important for HKi2 activity. The IC 50 was measured using a flow cytometry‐screening assay. >500 = no inhibition detected up to 500 μM concentration thus IC 50 >500 μM.

    Journal: FASEB BioAdvances

    Article Title: Inhibition of the HEG1–KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells

    doi: 10.1096/fba.2020-00141

    Figure Lengend Snippet: The aldehyde in position C1 and hydroxyl group in position C2 are important for HKi2 activity. The IC 50 was measured using a flow cytometry‐screening assay. >500 = no inhibition detected up to 500 μM concentration thus IC 50 >500 μM.

    Article Snippet: Assay plates were sampled using the HyperCyt TM high throughput flow cytometry platform (Intellicyt).

    Techniques: Activity Assay, Flow Cytometry, Screening Assay, Inhibition, Concentration Assay