Structured Review

Thermo Fisher edta
Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 672 article reviews
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edta - by Bioz Stars, 2020-01
90/100 stars

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Related Articles

Centrifugation:

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters. .. Dispersed islet cells were recovered by centrifugation in RPMI media containing 5.6mM glucose, 10% FBS, and 1% Penicillin/Streptomycin.

Immunocytochemistry:

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters. .. Air-dried slides were stored at -80C until use, thawed, and immediately fixed in 4% paraformaldehyde before immunocytochemistry.

Stable Transfection:

Article Title: Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes
Article Snippet: Epithelial cell monolayers were detached by one wash in PBS/0.5 mM EDTA followed by incubation in 0.05% trypsin/0.53 mM EDTA (Gibco). .. Cell lines stably expressing GFP-LC3 were generated by lentiviral infection with MOIs of 10–40.

Article Title: Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells ▿
Article Snippet: For fusion assays with polarized cells, MDCK cells stably expressing Fwt protein were seeded onto filter supports and were cultivated for 5 days. .. Since transfection of polarized cells is enhanced by a short treatment with trypsin or EDTA , 0.05% trypsin in 0.53 mM EDTA (Invitrogen) was added to the apical and basal chambers for 10 min to loosen the cell junctions.

Cytometry:

Article Title: AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway
Article Snippet: .. Flow cytometric analysis of cell differentiation To measure cell differentiation, THP-1 cells (1 × 106 ) were split into 35-mm dishes and treated with DMSO or 100 ng/ml PMA for 48 h. The cells were trypsinized, washed, and resuspended in cold PBS with 1 mM EDTA, 1% bovine serum albumin, and 10 mM sodium azide for 1 h. Before flow cytometric analysis, the cells were stained with PE-CD11b (12-0118-42) and APC-CD14 (17-0149-42) antibodies (eBioscience) for 30 min, washed using PBS with 1 mM EDTA and 1% bovine serum albumin, then subjected to flow cytometry using a BD Accuri™ C6 cytometer (BD Biosciences). .. Luciferase assay For the transcriptional activity assays, HEK 293T cells (2 × 104 ) were seeded in 48-well plates and co-transfected with the pGL3-p21 promoter or pGL3-KDM6B promoter reporter plasmid and the indicated DNA constructs using polyethylenimine (Polysciences), or treated with 0.1 or 0.3 μM alisertib, or treated with 2 or 5 μM GSK-J4, for 24 h. After transfection, the cells were collected and subjected to a luciferase assay (Promega).

Blocking Assay:

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: Wells were blocked for 30 min with 1% BSA to block nonspecific binding and washed 3× with serum-free DMEM prior to addition of cells. .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (#15575-020, ThermoFisher), to inhibit integrin activity, for 30 min in serum-free DMEM.

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: Wells were blocked for 30 min with 1% BSA to block nonspecific binding and washed 3× with serum-free DMEM prior to addition of cells. .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (Thermo), to inhibit integrin activity, for 30 min in serum-free DMEM.

Real-time Polymerase Chain Reaction:

Article Title: Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons
Article Snippet: Paragraph title: 4.5. Single-Cell Quantitative Real-Time PCR ... Cells were washed with 1X PBS, pH 7.4, supplemented with 3 mM EDTA, and enzymatically dissociated with 0.25% Trypsin supplemented with 0.5 mM EDTA (all Gibco).

Incubation:

Article Title: The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans
Article Snippet: C . elegans pellets were lysed in 20 mM Tris-HCl (pH 8.0), 170 mM KCl, 0.5% NP-40 (IGEPAL CA-360), 2 mM EDTA, with 40 μL of 100x Halt Protease & Phosphatase Inhibitor, 40 μL 100x EDTA (0.5 M) (Thermo #78446) per mL of lysis buffer. .. Lysates were prepared and quantified as above, and then 1.5 mg of lysate was precleared by the addition of 10 μL of normal rabbit serum (Invitrogen # 016101) and incubated with rotation for 1 h at 4°C, at which time the equivalent of 120 μL of drained Protein-A-Sepharose was added to immunoprecipitate non-specific interactions and incubation continued an additional 2 h. The proteins were eluted from the nonspecific pellet fraction with the addition of equal volume 2x SDS reducing buffer (1x SDS reducing buffer: 62.5 mM Tris (pH 6.8), 10% glycerol, 0.02% SDS, and 5% β-mercaptoethanol) and boiled for 5 min. Precleared lysate was evenly divided, antibodies were added, and lysates were incubated with rotation for 2 h at 4°C.

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (#15575-020, ThermoFisher), to inhibit integrin activity, for 30 min in serum-free DMEM. .. Cells were added in the presence or absence of 10 mM EDTA to ECM coated plates and incubated for 60 min at 37 °C.

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (Thermo), to inhibit integrin activity, for 30 min in serum-free DMEM. .. Cells were added in the presence or absence of 10 mM EDTA in serum-free DMEM with no additional sugar, 10 mM sucrose or 10 mM lactose to ECM coated plates and incubated for 60 min at 37 °C.

Article Title: Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer’s disease-linked presenilin 1 A246E mutation can be reversed with cAMP
Article Snippet: .. Cells were incubated at 37°C in 5.5% CO2 and sub-cultured by room temperature incubation in 0.53mM EDTA in HBSS (−Mg2+ /−Ca2+ ), followed by incubation with 0.05% trypsin/0.48mM EDTA (Life Technologies) at 37°C. ..

Article Title: Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes
Article Snippet: .. Epithelial cell monolayers were detached by one wash in PBS/0.5 mM EDTA followed by incubation in 0.05% trypsin/0.53 mM EDTA (Gibco). .. Cell lines stably expressing GFP-LC3 were generated by lentiviral infection with MOIs of 10–40.

Article Title: Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells ▿
Article Snippet: To visualize cell nuclei, cells were subsequently incubated with 4′,6-diamidino-2-phenylindole (DAPI). .. Since transfection of polarized cells is enhanced by a short treatment with trypsin or EDTA , 0.05% trypsin in 0.53 mM EDTA (Invitrogen) was added to the apical and basal chambers for 10 min to loosen the cell junctions.

Activity Assay:

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (#15575-020, ThermoFisher), to inhibit integrin activity, for 30 min in serum-free DMEM. .. Cells were added in the presence or absence of 10 mM EDTA to ECM coated plates and incubated for 60 min at 37 °C.

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (Thermo), to inhibit integrin activity, for 30 min in serum-free DMEM. .. Cells were added in the presence or absence of 10 mM EDTA in serum-free DMEM with no additional sugar, 10 mM sucrose or 10 mM lactose to ECM coated plates and incubated for 60 min at 37 °C.

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: Recombinant human defensin b1 protein (AbD Serotec) was purchased and biological activity was confirmed by the vendor as the ability to stimulate CD34+ dendritic cell migration. .. After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters.

Cell Culture:

Article Title: Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer’s disease-linked presenilin 1 A246E mutation can be reversed with cAMP
Article Snippet: This study used two distinct sets of control (CTRL) and PS1-fAD (A246E) human skin fibroblasts from the NIA Aging Cell Culture Repository (Coriell, Camden, NJ): cell numbers AG6840 and AG08170 were from two different PS1-fAD donors and termed “PS1-fAD” cells, while numbers AG07621 and AG07623 were the control cells from unaffected spouses of AD patients (termed “CTRL” cells). .. Cells were incubated at 37°C in 5.5% CO2 and sub-cultured by room temperature incubation in 0.53mM EDTA in HBSS (−Mg2+ /−Ca2+ ), followed by incubation with 0.05% trypsin/0.48mM EDTA (Life Technologies) at 37°C.

Article Title: Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons
Article Snippet: Single-Cell Quantitative Real-Time PCR Human myxoid liposarcoma cell line 402-91 was cultured in RPMI 1640 GlutaMAX medium supplemented with 5% fetal bovine serum, 50 U/mL penicillin and 50 mg/mL streptomycin (all Gibco, Grand Island, NY, USA) at 37 °C and 5% CO2 . .. Cells were washed with 1X PBS, pH 7.4, supplemented with 3 mM EDTA, and enzymatically dissociated with 0.25% Trypsin supplemented with 0.5 mM EDTA (all Gibco).

Article Title: Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes
Article Snippet: All epithelial cell lines were routinely cultured in DMEM with 10% fetal calf serum (FCS, Sigma), 2 mM glutamine, 110 μg/ml sodium pyruvate and 2 μg/ml gentamycin, except MLE-12, which was cultured in a 50:50 mix of DMEM:Ham’s F12, supplemented with 2% FCS, 5 μg/ml insulin, 10 μg/ml transferrin, 30 mM sodium selenite, 10 nM hydrocortisone, 10 nM beta-estradiol, 2 mM glutamine and 2 μg/ml gentamycin. .. Epithelial cell monolayers were detached by one wash in PBS/0.5 mM EDTA followed by incubation in 0.05% trypsin/0.53 mM EDTA (Gibco).

Expressing:

Article Title: Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes
Article Snippet: Epithelial cell monolayers were detached by one wash in PBS/0.5 mM EDTA followed by incubation in 0.05% trypsin/0.53 mM EDTA (Gibco). .. Cell lines stably expressing GFP-LC3 were generated by lentiviral infection with MOIs of 10–40.

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: Recombinant mouse Activin A protein was purchased from R & D Biosystems and biological activity was confirmed by the vendor as the ability to stimulate hemoglobin expression in K562 human chronic myologenous leukemia cells. .. After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters.

Article Title: Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells ▿
Article Snippet: For fusion assays with polarized cells, MDCK cells stably expressing Fwt protein were seeded onto filter supports and were cultivated for 5 days. .. Since transfection of polarized cells is enhanced by a short treatment with trypsin or EDTA , 0.05% trypsin in 0.53 mM EDTA (Invitrogen) was added to the apical and basal chambers for 10 min to loosen the cell junctions.

BIA-KA:

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: Western blotting Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). .. Tx-100 soluble cell lysates were subject to BCA protein assay (Pierce) and 30 μg protein/sample was separated by SDS-polyacrylamide gel electrophoresis and analyzed by western blotting with antibodies against the following targets: β2AR (Santa Cruz sc-569), α/β-Tubulin (Cell Signaling 2148 S), β-actin (Sigma-Aldrich A5316), GAPDH (ThermoFisher Scientific AM4300), and phosphorylated PKA substrate (Cell Signaling 9624).

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). .. Tx-100 soluble cell lysates were subject to BCA protein assay (Pierce) and 30 μg protein/sample was separated by SDS-polyacrylamide gel electrophoresis and analyzed by western blotting with antibodies against the following targets: β2AR (Santa Cruz sc-569), α/β-Tubulin (Cell Signaling 2148 S), β-actin (Sigma-Aldrich A5316), GAPDH (ThermoFisher Scientific AM4300), and phosphorylated PKA substrate (Cell Signaling 9624).

Western Blot:

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: .. Western blotting Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). ..

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: Paragraph title: Western blotting ... Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce).

Transfection:

Article Title: Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells ▿
Article Snippet: .. Since transfection of polarized cells is enhanced by a short treatment with trypsin or EDTA , 0.05% trypsin in 0.53 mM EDTA (Invitrogen) was added to the apical and basal chambers for 10 min to loosen the cell junctions. ..

Concentration Assay:

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: Final media concentration of 1%BSA and 0.5mM palmitate was used for islet cultures. .. After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters.

Protease Inhibitor:

Article Title: E-cadherin is required for intestinal morphogenesis in the mouse
Article Snippet: .. For whole cell extracts, cell pellets were lysed in buffer containing 0.5% NP-40, 50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, 250 mM NaCl, and HALT™ Protease Inhibitor cocktail without EDTA (Thermo Scientific, Rockford, IL). .. For nuclear extracts, cells were lysed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL), HALT™ Protease Inhibitor cocktail without EDTA (Thermo Scientific, Rockford, IL), and HALT™ Phosphatase Inhibitor cocktail (Thermo Scientific, Rockford, IL).

Infection:

Article Title: Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes
Article Snippet: Epithelial cell monolayers were detached by one wash in PBS/0.5 mM EDTA followed by incubation in 0.05% trypsin/0.53 mM EDTA (Gibco). .. Cell lines stably expressing GFP-LC3 were generated by lentiviral infection with MOIs of 10–40.

Cell Differentiation:

Article Title: AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway
Article Snippet: .. Flow cytometric analysis of cell differentiation To measure cell differentiation, THP-1 cells (1 × 106 ) were split into 35-mm dishes and treated with DMSO or 100 ng/ml PMA for 48 h. The cells were trypsinized, washed, and resuspended in cold PBS with 1 mM EDTA, 1% bovine serum albumin, and 10 mM sodium azide for 1 h. Before flow cytometric analysis, the cells were stained with PE-CD11b (12-0118-42) and APC-CD14 (17-0149-42) antibodies (eBioscience) for 30 min, washed using PBS with 1 mM EDTA and 1% bovine serum albumin, then subjected to flow cytometry using a BD Accuri™ C6 cytometer (BD Biosciences). .. Luciferase assay For the transcriptional activity assays, HEK 293T cells (2 × 104 ) were seeded in 48-well plates and co-transfected with the pGL3-p21 promoter or pGL3-KDM6B promoter reporter plasmid and the indicated DNA constructs using polyethylenimine (Polysciences), or treated with 0.1 or 0.3 μM alisertib, or treated with 2 or 5 μM GSK-J4, for 24 h. After transfection, the cells were collected and subjected to a luciferase assay (Promega).

Generated:

Article Title: Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes
Article Snippet: Epithelial cell monolayers were detached by one wash in PBS/0.5 mM EDTA followed by incubation in 0.05% trypsin/0.53 mM EDTA (Gibco). .. Cell lines stably expressing GFP-LC3 were generated by lentiviral infection with MOIs of 10–40.

Imaging:

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: Western blotting Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). .. Standard horseradish peroxidase secondary antibodies (1:10,000) and ECL reagent (ThermoFisher Scientific) were used for visualization with a charge-coupled device camera imaging system (UVP).

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). .. Standard horseradish peroxidase secondary antibodies (1:10,000) and ECL reagent (ThermoFisher Scientific) were used for visualization with a charge-coupled device camera imaging system (UVP).

Polymerase Chain Reaction:

Article Title: Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons
Article Snippet: Cells were washed with 1X PBS, pH 7.4, supplemented with 3 mM EDTA, and enzymatically dissociated with 0.25% Trypsin supplemented with 0.5 mM EDTA (all Gibco). .. Individual cells were then sorted into 96-well PCR plates, using a BD FACSAria II (BD Biosciences).

Injection:

Article Title: The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans
Article Snippet: C . elegans pellets were lysed in 20 mM Tris-HCl (pH 8.0), 170 mM KCl, 0.5% NP-40 (IGEPAL CA-360), 2 mM EDTA, with 40 μL of 100x Halt Protease & Phosphatase Inhibitor, 40 μL 100x EDTA (0.5 M) (Thermo #78446) per mL of lysis buffer. .. The antibodies used in various immunoprecipitations were as follows: for HSF-1 1 μL of HSF-1 immune serum, and for negative controls either 1 μL of commercially available NRS (Invitrogen # 016101) or pre-immune serum from the rabbit prior to injection with recombinant C . elegans recombinant HSF-1.

Binding Assay:

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: Wells were blocked for 30 min with 1% BSA to block nonspecific binding and washed 3× with serum-free DMEM prior to addition of cells. .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (#15575-020, ThermoFisher), to inhibit integrin activity, for 30 min in serum-free DMEM.

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: Wells were blocked for 30 min with 1% BSA to block nonspecific binding and washed 3× with serum-free DMEM prior to addition of cells. .. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (Thermo), to inhibit integrin activity, for 30 min in serum-free DMEM.

Nucleic Acid Electrophoresis:

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: Western blotting Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). .. Tx-100 soluble cell lysates were subject to BCA protein assay (Pierce) and 30 μg protein/sample was separated by SDS-polyacrylamide gel electrophoresis and analyzed by western blotting with antibodies against the following targets: β2AR (Santa Cruz sc-569), α/β-Tubulin (Cell Signaling 2148 S), β-actin (Sigma-Aldrich A5316), GAPDH (ThermoFisher Scientific AM4300), and phosphorylated PKA substrate (Cell Signaling 9624).

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). .. Tx-100 soluble cell lysates were subject to BCA protein assay (Pierce) and 30 μg protein/sample was separated by SDS-polyacrylamide gel electrophoresis and analyzed by western blotting with antibodies against the following targets: β2AR (Santa Cruz sc-569), α/β-Tubulin (Cell Signaling 2148 S), β-actin (Sigma-Aldrich A5316), GAPDH (ThermoFisher Scientific AM4300), and phosphorylated PKA substrate (Cell Signaling 9624).

Mutagenesis:

Article Title: E-cadherin is required for intestinal morphogenesis in the mouse
Article Snippet: Protein was extracted from tissue or isolated epithelial cells of control and E-cadherin mutant E18.5 small intestines. .. For whole cell extracts, cell pellets were lysed in buffer containing 0.5% NP-40, 50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, 250 mM NaCl, and HALT™ Protease Inhibitor cocktail without EDTA (Thermo Scientific, Rockford, IL).

Article Title: Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells ▿
Article Snippet: Twenty-four hours later, cells were transfected with plasmids encoding either wild-type or mutant NiV G. Cell fusion was analyzed at 24 h posttransfection (p.t.) by immunostaining with the G-specific monoclonal antibody mab26 and Alexa Fluor 568-conjugated secondary antibodies. .. Since transfection of polarized cells is enhanced by a short treatment with trypsin or EDTA , 0.05% trypsin in 0.53 mM EDTA (Invitrogen) was added to the apical and basal chambers for 10 min to loosen the cell junctions.

Isolation:

Article Title: E-cadherin is required for intestinal morphogenesis in the mouse
Article Snippet: Protein was extracted from tissue or isolated epithelial cells of control and E-cadherin mutant E18.5 small intestines. .. For whole cell extracts, cell pellets were lysed in buffer containing 0.5% NP-40, 50 mM Tris-HCl, pH 8.0, 10% glycerol, 0.1 mM EDTA, 250 mM NaCl, and HALT™ Protease Inhibitor cocktail without EDTA (Thermo Scientific, Rockford, IL).

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: Paragraph title: Islet isolation and Cytospin assay ... After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters.

Flow Cytometry:

Article Title: AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway
Article Snippet: .. Flow cytometric analysis of cell differentiation To measure cell differentiation, THP-1 cells (1 × 106 ) were split into 35-mm dishes and treated with DMSO or 100 ng/ml PMA for 48 h. The cells were trypsinized, washed, and resuspended in cold PBS with 1 mM EDTA, 1% bovine serum albumin, and 10 mM sodium azide for 1 h. Before flow cytometric analysis, the cells were stained with PE-CD11b (12-0118-42) and APC-CD14 (17-0149-42) antibodies (eBioscience) for 30 min, washed using PBS with 1 mM EDTA and 1% bovine serum albumin, then subjected to flow cytometry using a BD Accuri™ C6 cytometer (BD Biosciences). .. Luciferase assay For the transcriptional activity assays, HEK 293T cells (2 × 104 ) were seeded in 48-well plates and co-transfected with the pGL3-p21 promoter or pGL3-KDM6B promoter reporter plasmid and the indicated DNA constructs using polyethylenimine (Polysciences), or treated with 0.1 or 0.3 μM alisertib, or treated with 2 or 5 μM GSK-J4, for 24 h. After transfection, the cells were collected and subjected to a luciferase assay (Promega).

Immunostaining:

Article Title: Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells ▿
Article Snippet: Twenty-four hours later, cells were transfected with plasmids encoding either wild-type or mutant NiV G. Cell fusion was analyzed at 24 h posttransfection (p.t.) by immunostaining with the G-specific monoclonal antibody mab26 and Alexa Fluor 568-conjugated secondary antibodies. .. Since transfection of polarized cells is enhanced by a short treatment with trypsin or EDTA , 0.05% trypsin in 0.53 mM EDTA (Invitrogen) was added to the apical and basal chambers for 10 min to loosen the cell junctions.

Lysis:

Article Title: The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans
Article Snippet: .. C . elegans pellets were lysed in 20 mM Tris-HCl (pH 8.0), 170 mM KCl, 0.5% NP-40 (IGEPAL CA-360), 2 mM EDTA, with 40 μL of 100x Halt Protease & Phosphatase Inhibitor, 40 μL 100x EDTA (0.5 M) (Thermo #78446) per mL of lysis buffer. .. Lysates were prepared and quantified as above, and then 1.5 mg of lysate was precleared by the addition of 10 μL of normal rabbit serum (Invitrogen # 016101) and incubated with rotation for 1 h at 4°C, at which time the equivalent of 120 μL of drained Protein-A-Sepharose was added to immunoprecipitate non-specific interactions and incubation continued an additional 2 h. The proteins were eluted from the nonspecific pellet fraction with the addition of equal volume 2x SDS reducing buffer (1x SDS reducing buffer: 62.5 mM Tris (pH 6.8), 10% glycerol, 0.02% SDS, and 5% β-mercaptoethanol) and boiled for 5 min. Precleared lysate was evenly divided, antibodies were added, and lysates were incubated with rotation for 2 h at 4°C.

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: .. Western blotting Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). ..

Article Title: β2-Adrenergic receptor modulates mitochondrial metabolism and disease progression in recurrent/metastatic HPV(+) HNSCC
Article Snippet: .. Cells were grown to 80% confluence and collected in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mN Na3 VO4 , 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1% Triton X-100 [Tx-100], 17.4 μg/mL phenylmethylsulfonylfluoride, 1 × HALT with EDTA), 1% Tx-100, and HALT with EDTA (Pierce). ..

Recombinant:

Article Title: The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans
Article Snippet: C . elegans pellets were lysed in 20 mM Tris-HCl (pH 8.0), 170 mM KCl, 0.5% NP-40 (IGEPAL CA-360), 2 mM EDTA, with 40 μL of 100x Halt Protease & Phosphatase Inhibitor, 40 μL 100x EDTA (0.5 M) (Thermo #78446) per mL of lysis buffer. .. The antibodies used in various immunoprecipitations were as follows: for HSF-1 1 μL of HSF-1 immune serum, and for negative controls either 1 μL of commercially available NRS (Invitrogen # 016101) or pre-immune serum from the rabbit prior to injection with recombinant C . elegans recombinant HSF-1.

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: Recombinant human defensin b1 protein (AbD Serotec) was purchased and biological activity was confirmed by the vendor as the ability to stimulate CD34+ dendritic cell migration. .. After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters.

Immunoprecipitation:

Article Title: The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in Caenorhabditis elegans
Article Snippet: Paragraph title: Immunoprecipitation ... C . elegans pellets were lysed in 20 mM Tris-HCl (pH 8.0), 170 mM KCl, 0.5% NP-40 (IGEPAL CA-360), 2 mM EDTA, with 40 μL of 100x Halt Protease & Phosphatase Inhibitor, 40 μL 100x EDTA (0.5 M) (Thermo #78446) per mL of lysis buffer.

Migration:

Article Title: Interrupted Glucagon Signaling Reveals Hepatic-α-Cell Axis and Role for L-Glutamine in α-Cell Proliferation
Article Snippet: Recombinant human defensin b1 protein (AbD Serotec) was purchased and biological activity was confirmed by the vendor as the ability to stimulate CD34+ dendritic cell migration. .. After culture, islets were washed in 2mM EDTA and dispersed in 0.025% Trypsin-2 mM EDTA (Gibco Life Technologies or HyClone GE Healthcare) for 5-10 minutes by gentle pipetting to obtain single or very small cell clusters.

Staining:

Article Title: Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells ▿
Article Snippet: Since transfection of polarized cells is enhanced by a short treatment with trypsin or EDTA , 0.05% trypsin in 0.53 mM EDTA (Invitrogen) was added to the apical and basal chambers for 10 min to loosen the cell junctions. .. Cell fusion was monitored by immunostaining at 48 h after transfection with the G-specific monoclonal antibody (mab26), and cell junctions were visualized by E-cadherin staining as described above.

Article Title: AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway
Article Snippet: .. Flow cytometric analysis of cell differentiation To measure cell differentiation, THP-1 cells (1 × 106 ) were split into 35-mm dishes and treated with DMSO or 100 ng/ml PMA for 48 h. The cells were trypsinized, washed, and resuspended in cold PBS with 1 mM EDTA, 1% bovine serum albumin, and 10 mM sodium azide for 1 h. Before flow cytometric analysis, the cells were stained with PE-CD11b (12-0118-42) and APC-CD14 (17-0149-42) antibodies (eBioscience) for 30 min, washed using PBS with 1 mM EDTA and 1% bovine serum albumin, then subjected to flow cytometry using a BD Accuri™ C6 cytometer (BD Biosciences). .. Luciferase assay For the transcriptional activity assays, HEK 293T cells (2 × 104 ) were seeded in 48-well plates and co-transfected with the pGL3-p21 promoter or pGL3-KDM6B promoter reporter plasmid and the indicated DNA constructs using polyethylenimine (Polysciences), or treated with 0.1 or 0.3 μM alisertib, or treated with 2 or 5 μM GSK-J4, for 24 h. After transfection, the cells were collected and subjected to a luciferase assay (Promega).

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