calcium chloride dihydrate  (Millipore)


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    Name:
    Calcium chloride dihydrate
    Description:

    Catalog Number:
    c2536
    Price:
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    Structured Review

    Millipore calcium chloride dihydrate
    Calcium chloride dihydrate

    https://www.bioz.com/result/calcium chloride dihydrate/product/Millipore
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    calcium chloride dihydrate - by Bioz Stars, 2020-02
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    Related Articles

    Stable Transfection:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: For APC ANXA5/annexin V (ex/em maxima 650/660)-PI measurements, the cell pellets were resuspended in 50 µl FACS buffer (10 mM HEPES [AppliChem, 7365–45-9], 140 mM NaCl [Sigma-Aldrich, 31,434], 5 mM CaCl2 [Sigma-Aldrich, C3306], pH 7.4) with 0.8 µl APC ANXA5/annexin V and 0.8 µg/mL PI. .. For the mt-mKeima assay, 100,000 cells stably expressing mKeima were seeded into 12-well plates one day before the measurement.

    Cytometry:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: Paragraph title: Flow cytometry ... For APC ANXA5/annexin V (ex/em maxima 650/660)-PI measurements, the cell pellets were resuspended in 50 µl FACS buffer (10 mM HEPES [AppliChem, 7365–45-9], 140 mM NaCl [Sigma-Aldrich, 31,434], 5 mM CaCl2 [Sigma-Aldrich, C3306], pH 7.4) with 0.8 µl APC ANXA5/annexin V and 0.8 µg/mL PI.

    Construct:

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: For overexpression studies, a pcDNA3.1 (−) construct containing the full murine Tspo cDNA ORF, a generous gift from Prof. V. Papadopoulos (McGill University) was transfected; the empty vector was used as a control in this instance. .. Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Synergy Between Gαz Deficiency and GLP-1 Analog Treatment in Preserving Functional β-Cell Mass in Experimental Diabetes
    Article Snippet: Sodium chloride (S9888), potassium chloride (P3911), magnesium sulfate heptahydrate (M9397), potassium phosphate monobasic (P0662), sodium bicarbonate (S6014), HEPES (H3375), calcium chloride dehydrate (C3881), and RIA-grade bovine serum albumin (A7888), STZ (S0130), and Ex4 (E7144) were purchased from Sigma-Aldrich. .. Antiinsulin ELISA antibodies (insulin + proinsulin antibody, 10R-I136a; insulin + proinsulin antibody, biotinylated, 61E-I136bBT) were from Fitzgerald Industries.

    Incubation:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: DTT (1,4-dithiothreitol; AppliChem, 28,194,437) was added to a final concentration of 5 mM and the samples were incubated for 25 min at 56°C, followed by the addition of iodoacetamide (Sigma-Aldrich, I1149) to 14 mM final concentration and a further incubation for 30 min at room temperature in the dark. .. Samples were diluted 1:5 in 25 mM Tris-HCl (Sigma-Aldrich, T1530), pH 8.2, and CaCl2 (Sigma-Aldrich, C3306) was added to a final concentration of 1 mM.

    Article Title: Autophagy mediates phosphatidylserine exposure and phagosome degradation during apoptosis through specific functions of GABARAP/LGG-1 and LC3/LGG-2
    Article Snippet: Nematode strains were grown on nematode growth media (NGM) plates (50 mM NaCl [Sigma-Aldrich, 60,142], 0.25% bactopeptone [Becton-Dickinson, 211677], 5 µg/ml cholesterol [Sigma-Aldrich, C8667], 2% bacto agar [Becton-Dickinson, 214010] autoclaved and supplemented with 1 mM CaCl2 [Sigma-Aldrich, C3306], 1 mM MgSO4 [Sigma-Aldrich, M5921], 20 mM KH2 PO4 [Sigma-Aldrich, P5655] 1 M, 3.3 mM K2 HPO4 [Sigma-Aldrich, 60356]) and fed with Escherichia coli strain OP50 (Caenorhabditis Genetic Center) [ ]. .. To score ACs labelled with ANXV::GFP or GFP::LACTC1C2, embryos were grown at 20°C and then incubated at 33°C for 45 min followed by a recovery at 20°C for 2 h. The quantification is the ratio between the number of DIC corpses and the fluorescent rings regardless of cell morphology.

    Transfection:

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: Paragraph title: Cell culture, transfections, and gene expression ... Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    Expressing:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: For APC ANXA5/annexin V (ex/em maxima 650/660)-PI measurements, the cell pellets were resuspended in 50 µl FACS buffer (10 mM HEPES [AppliChem, 7365–45-9], 140 mM NaCl [Sigma-Aldrich, 31,434], 5 mM CaCl2 [Sigma-Aldrich, C3306], pH 7.4) with 0.8 µl APC ANXA5/annexin V and 0.8 µg/mL PI. .. For the mt-mKeima assay, 100,000 cells stably expressing mKeima were seeded into 12-well plates one day before the measurement.

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling. .. Heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich cat. no. F4135–500ML) Normal Goat Serum (NGS, Sigma-Aldrich cat. no. G6767–500ML) Triton X-100 (Sigma-Aldrich cat. no. T8787–100ML) Hoechst (Santa Cruz cat. no. 33258) Oligonucleotide pool (CustomArray) Primers (Sigma-Aldrich) dNTPs (New England Biolabs cat. no. N0447S) Agarose (Invitrogen cat. no. 16500–500) Sybr Safe (Invitrogen cat. no. S33102) Alexa488-conjugated goat anti-mouse (Life Sciences cat. no. ) Alexa594-conjugated goat anti-rabbit (Life Sciences cat. no. ) Mouse anti-FLAG (Sigma-Aldrich cat. no. F1804–50UG) Counterstain antibody not made in a mouse DG-52 mouse anti-SAG1 Alexa-488 labeling kit (Life Technology ) Midiprep kit (Macherey-Nagel cat. no. 740412.50) Miniprep kit (Zymo cat. no. D4054) DNeasy DNA extraction kit (Qiagen cat. no. 69504) QIAquick gel extraction kit (Qiagen cat. no. 28706) Gibson Assembly (New England Biolabs cat. no. E2611L) E. cloni 10G Supreme electrocompetent cells (VWR cat. no. 95024–012) AseI and associated NEBuffer 3.1 (New England Biolabs cat. no. R0526L) DNaseI (Sigma-Aldrich cat. no. DN25–100MG) Trypsin (Worthington cat. no. ) iProof and HF buffer (BioRad cat. no. 422840) Q5 DNA Polymerase (NEB cat. no. M0491L) BsaI and associated Buffer G (Thermo Scientific cat. no. ER0291) Calf Intestinal Phosphatase (CIP, New England Biolabs cat. no. M0290S) HFF cells (American Type Culture Collection cat. no. SCRC-1041) RH/Cas9 (American Type Culture Collection or by request) CAUTION Check for Cas9 expression and function before starting a screen by following steps 1–25 in the protocol.

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: Paragraph title: Cell culture, transfections, and gene expression ... Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    BIA-KA:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: Supernatant was transferred into a new tube and protein concentration was determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, 23,225) according to the manufacturer’s manual. .. Samples were diluted 1:5 in 25 mM Tris-HCl (Sigma-Aldrich, T1530), pH 8.2, and CaCl2 (Sigma-Aldrich, C3306) was added to a final concentration of 1 mM.

    Modification:

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: .. Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4. .. Cells were fixed in ice-cold acetone for 10 min at −20°C, followed by 3 × 5 min washes in phosphate-buffered saline (PBS) (Sigma-Aldrich, P4417).

    Over Expression:

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: For overexpression studies, a pcDNA3.1 (−) construct containing the full murine Tspo cDNA ORF, a generous gift from Prof. V. Papadopoulos (McGill University) was transfected; the empty vector was used as a control in this instance. .. Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    High Performance Liquid Chromatography:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Blunt needles (SAI Infusion cat. no. B27–50) can be used as an alternative. pU6-SAG1 (Addgene cat. no. 80322) Lourido Toxoplasma CRISPR Library V1 (Addgene cat. no. 80636) pU6-DHFR (Addgene cat. no. 80329) pCas9-CAT (Addgene cat. no. 80323) pU6-Decoy (Addgene cat. no. 80324) Ampicillin (Sigma-Aldrich-Aldrich cat. no. A0166–25G) Glycerol (EMD cat. no. GX0185–6) Methanol-Free Formaldehyde (Thermo Scientific cat. no. 28908) Prolong Gold (Invitrogen cat. no. P1044) Molecular biology-grade water (e.g., from an EMD Millipore Milii-Q Integral System, Fisher Scientific cat. no. ZRXQ003US) Gentamicin (Gibco cat. no. 15710–064) EGTA (Sigma-Aldrich cat. no. E4378–500G) HEPES (Sigma-Aldrich cat. no. 3375–1KG) KH2 PO4 (Sigma-Aldrich cat. no. P5655) K2 HPO4 (Sigma-Aldrich cat. no. 1551128) KCl (Fisher Scientific cat. no. SP138–500) MgCl2 (Mallinckrodt cat. no. 5958) Methanol for HPLC gradient analysis (Millipore, cat. No. 67–56-1) EDTA (Amresco cat. no. 0322–500G) Tris base (Sigma-Aldrich cat. no. TRIS-RO) Glacial acetic acid (Spectrum cat. no. A1010) CAUTION Glacial acetic acid is corrosive; avoid contact with eyes, skin, and clothing; wear protective gloves while handling. .. Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling.

    Flow Cytometry:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: Paragraph title: Flow cytometry ... For APC ANXA5/annexin V (ex/em maxima 650/660)-PI measurements, the cell pellets were resuspended in 50 µl FACS buffer (10 mM HEPES [AppliChem, 7365–45-9], 140 mM NaCl [Sigma-Aldrich, 31,434], 5 mM CaCl2 [Sigma-Aldrich, C3306], pH 7.4) with 0.8 µl APC ANXA5/annexin V and 0.8 µg/mL PI.

    Transferring:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Eppendorf tubes (Axygen cat. no. MCT-150-C) 50 ml tubes (Corning cat. no. 430828) 15 ml tubes (Corning cat. no. 430052) Pipettes (Fisher Scientific cat. no. F144802G and F167300G) Pipette tips with filters (USA Scientific cat. no. 1120–1810, 1120–8810, 1122–1830 and 1120–3810) Sterile Acrodisc® Syringe Filters with HT Tuffryn® Membrane 0.2 μm, 25 mm (Pall Life Sciences cat. no. 4192). .. Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling.

    Cell Culture:

    Article Title: Differential Glucose Requirement in Skin Homeostasis and Injury Identifies a Therapeutic Target for Psoriasis
    Article Snippet: Keratinocyte Culture and Treatments HEK001 (ATCC CRL-2404) and SCCT8 (obtained from Andrew South) were cultured according to instructions. .. The cell solution was filtered the cells with a 70 µM cell strainer, centrifuged at 1000rpm for 4 min, and resuspended in KSFM medium (Invitrogen, 37010022) supplemented with 0.05 mM CaCl2 (Sigma, C7902), 0.05 µg/ml Hydrocortisone (Sigma, H0888), 5 ng/ml EGF (Invitrogen, 10450-013), 7.5 µg/ml Bovine Pituitary Extract (Invitrogen, 13028-014), 0.5 µg/ml insulin (Sigma, I9278), 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL of Amphotericin B (Gibco, 15240062).

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: Paragraph title: Cell culture, transfections, and gene expression ... Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    other:

    Article Title: Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles
    Article Snippet: Reagents Hepes (H3375), ascorbic acid (A5960), CaCl2 (C5080), and BSA (A8022) were purchased from Sigma-Aldrich.

    Article Title: Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig
    Article Snippet: PVA-TL-HEPES: 6.663 g NaCl (Sigma-S-5886), 0.239 g KCl (Sigma, P4504), 0.168 g NaHCO3 (Sigma, S8875), 0.041 g NaH2 PO4 (Sigma, S5011), 0.102 g MgCl2 .6H2 O (Sigma, M0250), 2.383 g HEPES (Sigma, H3784), 0.065 g penicillin (Sigma, P3032), 0.010 g Phenol Red (Sigma, 5,530), 0.294 g CaCl2 .2H2 O (Sigma,C7902), 0.1 g polyvinyl alcohol (PVA, Sigma, P8136), 2.186 g sorbitol (Sigma, S1876), 0.025 g Gentamicin (Gibco, 15710-064), 0.022 g sodium pyruvate (Sigma, P4562), 998 ml Milli Q H2 O and 1.868 ml Na Lactate (Sigma, L7900), adjusting pH to 7.2−7.4 and osmotic pressure to 295−310 mOsm.

    Article Title: High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers
    Article Snippet: Sodium chloride, NaCl (e.g. Sigma-Aldrich, Ref# S3014, USA) Potassium chloride, KCl (e.g. Sigma-Aldrich, Ref# P9333, USA) Sodium Phosphate monobasic monohydrate, NaH2 PO4 •H2 O (e.g. Fisher, Ref# S369, USA) Sodium phosphate dibasic, anhydrous, Na2 HPO4 (e.g. Acros Organics, Ref#42437-5000, USA) HEPES (e.g. Sigma-Aldrich, Ref# H4034, USA) Sodium bicarbonate, NaHCO3 (e.g. Fisher, Ref# S233, USA) EGTA (e.g. Sigma-Aldrich, Ref# E4378, USA) D-(+)-Glucose (e.g. Sigma-Aldrich, Ref# G8270, USA) Calcium chloride dihydrate, CaCl2 •H2 O (e.g. Sigma-Aldrich, Ref# C7902, USA) Magnesium chloride hexahydrate, MgCl2 •6H2 O (e.g. Malinckrodt Chemicals, Ref# 5958, UK) Magnesium sulphate heptahydrate, MgSO4 •7H2 O (e.g. Sigma-Aldrich, Ref#230391, USA) Potassium phosphate monobasic, KH2 PO4 (e.g. Malinckrodt Chemicals, Ref# 7100, UK) Optional: Gey´s Balanced Salt Solution B (GBSS/B) (Sigma-Aldrich, Ref# G9779, USA) ddH2 O

    Article Title: Cellular Fura-2 Manganese Extraction Assay (CFMEA)
    Article Snippet: 2H2 O (Catalog # C7902; Sigma) 1 mM MgSO4 .

    Gel Extraction:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling. .. Heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich cat. no. F4135–500ML) Normal Goat Serum (NGS, Sigma-Aldrich cat. no. G6767–500ML) Triton X-100 (Sigma-Aldrich cat. no. T8787–100ML) Hoechst (Santa Cruz cat. no. 33258) Oligonucleotide pool (CustomArray) Primers (Sigma-Aldrich) dNTPs (New England Biolabs cat. no. N0447S) Agarose (Invitrogen cat. no. 16500–500) Sybr Safe (Invitrogen cat. no. S33102) Alexa488-conjugated goat anti-mouse (Life Sciences cat. no. ) Alexa594-conjugated goat anti-rabbit (Life Sciences cat. no. ) Mouse anti-FLAG (Sigma-Aldrich cat. no. F1804–50UG) Counterstain antibody not made in a mouse DG-52 mouse anti-SAG1 Alexa-488 labeling kit (Life Technology ) Midiprep kit (Macherey-Nagel cat. no. 740412.50) Miniprep kit (Zymo cat. no. D4054) DNeasy DNA extraction kit (Qiagen cat. no. 69504) QIAquick gel extraction kit (Qiagen cat. no. 28706) Gibson Assembly (New England Biolabs cat. no. E2611L) E. cloni 10G Supreme electrocompetent cells (VWR cat. no. 95024–012) AseI and associated NEBuffer 3.1 (New England Biolabs cat. no. R0526L) DNaseI (Sigma-Aldrich cat. no. DN25–100MG) Trypsin (Worthington cat. no. ) iProof and HF buffer (BioRad cat. no. 422840) Q5 DNA Polymerase (NEB cat. no. M0491L) BsaI and associated Buffer G (Thermo Scientific cat. no. ER0291) Calf Intestinal Phosphatase (CIP, New England Biolabs cat. no. M0290S) HFF cells (American Type Culture Collection cat. no. SCRC-1041) RH/Cas9 (American Type Culture Collection or by request) CAUTION Check for Cas9 expression and function before starting a screen by following steps 1–25 in the protocol.

    Protein Concentration:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: Differentially AT 101-treated and SILAC-labelled U87MG cells were mixed at equal ratio based on protein concentration. .. Samples were diluted 1:5 in 25 mM Tris-HCl (Sigma-Aldrich, T1530), pH 8.2, and CaCl2 (Sigma-Aldrich, C3306) was added to a final concentration of 1 mM.

    Polymerase Chain Reaction:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Petri dishes (Falcon cat. no. 351029) Culture tubes (Corning cat. no. 352051) 24-well tissue culture dishes (Costar cat. no. 3524) Parafilm (VWR cat. no. 291–1214) T12.5 tissue culture flasks (Falcon cat. no. 353018) 15 cm tissue culture dishes (Falcon cat. no. 353025) Cell scrapers (Costar cat. no. 3010) PCR tubes (BioRad cat. no. TF102010) Cryotubes (Thermo Scientific cat. no. 5000–0012) 27½ -gauge needles (BD cat. no. 305109) CAUTION Exercise extreme care when handling live T. gondii with sharps. .. Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling.

    DNA Extraction:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling. .. Heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich cat. no. F4135–500ML) Normal Goat Serum (NGS, Sigma-Aldrich cat. no. G6767–500ML) Triton X-100 (Sigma-Aldrich cat. no. T8787–100ML) Hoechst (Santa Cruz cat. no. 33258) Oligonucleotide pool (CustomArray) Primers (Sigma-Aldrich) dNTPs (New England Biolabs cat. no. N0447S) Agarose (Invitrogen cat. no. 16500–500) Sybr Safe (Invitrogen cat. no. S33102) Alexa488-conjugated goat anti-mouse (Life Sciences cat. no. ) Alexa594-conjugated goat anti-rabbit (Life Sciences cat. no. ) Mouse anti-FLAG (Sigma-Aldrich cat. no. F1804–50UG) Counterstain antibody not made in a mouse DG-52 mouse anti-SAG1 Alexa-488 labeling kit (Life Technology ) Midiprep kit (Macherey-Nagel cat. no. 740412.50) Miniprep kit (Zymo cat. no. D4054) DNeasy DNA extraction kit (Qiagen cat. no. 69504) QIAquick gel extraction kit (Qiagen cat. no. 28706) Gibson Assembly (New England Biolabs cat. no. E2611L) E. cloni 10G Supreme electrocompetent cells (VWR cat. no. 95024–012) AseI and associated NEBuffer 3.1 (New England Biolabs cat. no. R0526L) DNaseI (Sigma-Aldrich cat. no. DN25–100MG) Trypsin (Worthington cat. no. ) iProof and HF buffer (BioRad cat. no. 422840) Q5 DNA Polymerase (NEB cat. no. M0491L) BsaI and associated Buffer G (Thermo Scientific cat. no. ER0291) Calf Intestinal Phosphatase (CIP, New England Biolabs cat. no. M0290S) HFF cells (American Type Culture Collection cat. no. SCRC-1041) RH/Cas9 (American Type Culture Collection or by request) CAUTION Check for Cas9 expression and function before starting a screen by following steps 1–25 in the protocol.

    Isolation:

    Article Title: Differential Glucose Requirement in Skin Homeostasis and Injury Identifies a Therapeutic Target for Psoriasis
    Article Snippet: Human and mouse primary keratinocytes were isolated by dispase digestion . .. The cell solution was filtered the cells with a 70 µM cell strainer, centrifuged at 1000rpm for 4 min, and resuspended in KSFM medium (Invitrogen, 37010022) supplemented with 0.05 mM CaCl2 (Sigma, C7902), 0.05 µg/ml Hydrocortisone (Sigma, H0888), 5 ng/ml EGF (Invitrogen, 10450-013), 7.5 µg/ml Bovine Pituitary Extract (Invitrogen, 13028-014), 0.5 µg/ml insulin (Sigma, I9278), 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL of Amphotericin B (Gibco, 15240062).

    Immunohistochemistry:

    Article Title: On-slide tissue digestion for mass spectrometry based glycomic and proteomic profiling
    Article Snippet: Reagents • Hydrophobic IHC Pap pen (SuperHT pen, Biotium) • Ethyl alcohol, 200 proof, (BP2818, Fisher Scientific) CAUTION flammable liquid. .. Wear gloves and safety goggles while handling • Ammonium acetate(A7330-500 g, Sigma Aldrich), • Ammonium bicarbonate(A6141, Sigma Aldrich) • Tris-HCl(T3253, Sigma, pH 8), • Calcium Chloride(C5080, Sigma Aldrich), • Ammonium hydroxide, 25–30%(320145, Sigma Aldrich) CAUTION corrosive and an irritant by vapor or ingestion.

    Labeling:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling. .. Heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich cat. no. F4135–500ML) Normal Goat Serum (NGS, Sigma-Aldrich cat. no. G6767–500ML) Triton X-100 (Sigma-Aldrich cat. no. T8787–100ML) Hoechst (Santa Cruz cat. no. 33258) Oligonucleotide pool (CustomArray) Primers (Sigma-Aldrich) dNTPs (New England Biolabs cat. no. N0447S) Agarose (Invitrogen cat. no. 16500–500) Sybr Safe (Invitrogen cat. no. S33102) Alexa488-conjugated goat anti-mouse (Life Sciences cat. no. ) Alexa594-conjugated goat anti-rabbit (Life Sciences cat. no. ) Mouse anti-FLAG (Sigma-Aldrich cat. no. F1804–50UG) Counterstain antibody not made in a mouse DG-52 mouse anti-SAG1 Alexa-488 labeling kit (Life Technology ) Midiprep kit (Macherey-Nagel cat. no. 740412.50) Miniprep kit (Zymo cat. no. D4054) DNeasy DNA extraction kit (Qiagen cat. no. 69504) QIAquick gel extraction kit (Qiagen cat. no. 28706) Gibson Assembly (New England Biolabs cat. no. E2611L) E. cloni 10G Supreme electrocompetent cells (VWR cat. no. 95024–012) AseI and associated NEBuffer 3.1 (New England Biolabs cat. no. R0526L) DNaseI (Sigma-Aldrich cat. no. DN25–100MG) Trypsin (Worthington cat. no. ) iProof and HF buffer (BioRad cat. no. 422840) Q5 DNA Polymerase (NEB cat. no. M0491L) BsaI and associated Buffer G (Thermo Scientific cat. no. ER0291) Calf Intestinal Phosphatase (CIP, New England Biolabs cat. no. M0290S) HFF cells (American Type Culture Collection cat. no. SCRC-1041) RH/Cas9 (American Type Culture Collection or by request) CAUTION Check for Cas9 expression and function before starting a screen by following steps 1–25 in the protocol.

    Sequencing:

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: As a control for these studies a nonsilencing siRNA sequence (AllStars Negative Control, Qiagen, 1027281) was transfected. .. Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    CRISPR:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Blunt needles (SAI Infusion cat. no. B27–50) can be used as an alternative. pU6-SAG1 (Addgene cat. no. 80322) Lourido Toxoplasma CRISPR Library V1 (Addgene cat. no. 80636) pU6-DHFR (Addgene cat. no. 80329) pCas9-CAT (Addgene cat. no. 80323) pU6-Decoy (Addgene cat. no. 80324) Ampicillin (Sigma-Aldrich-Aldrich cat. no. A0166–25G) Glycerol (EMD cat. no. GX0185–6) Methanol-Free Formaldehyde (Thermo Scientific cat. no. 28908) Prolong Gold (Invitrogen cat. no. P1044) Molecular biology-grade water (e.g., from an EMD Millipore Milii-Q Integral System, Fisher Scientific cat. no. ZRXQ003US) Gentamicin (Gibco cat. no. 15710–064) EGTA (Sigma-Aldrich cat. no. E4378–500G) HEPES (Sigma-Aldrich cat. no. 3375–1KG) KH2 PO4 (Sigma-Aldrich cat. no. P5655) K2 HPO4 (Sigma-Aldrich cat. no. 1551128) KCl (Fisher Scientific cat. no. SP138–500) MgCl2 (Mallinckrodt cat. no. 5958) Methanol for HPLC gradient analysis (Millipore, cat. No. 67–56-1) EDTA (Amresco cat. no. 0322–500G) Tris base (Sigma-Aldrich cat. no. TRIS-RO) Glacial acetic acid (Spectrum cat. no. A1010) CAUTION Glacial acetic acid is corrosive; avoid contact with eyes, skin, and clothing; wear protective gloves while handling. .. Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling.

    Plasmid Preparation:

    Article Title: An Efficient Protocol for Model Legume Root Protoplast Isolation and Transformation
    Article Snippet: Co., Japan); simple • D-Sorbitol (Sigma, cat. no. S3889); simple • Potassium chloride (KCl; Sigma-Aldrich, cat. no. P3911); simple • Calcium chloride dehydrate (CaCl2 ; Sigma, cat. no. C7902); simple • Sodium chloride (NaCl; Sigma-Aldrich, cat. no. S9888); simple • Magnesium chloride hexa-hydrate (MgCl2 . .. 6 H2O; Sigma-Aldrich, cat. no. 9272); simple • BSA (Shanghai Bo AO Biotech Co., Ltd., China); simple • MES hydrate (Sigma, cat. no. M8250); simple • Cell-wall degrading enzyme mix viscozyme (Sigma, cat. no. V2010); simple • PEG4000 (Sigma-Aldrich, cat. no. 81240); simple • Plasmid Maxi Kit (QIAGEN, cat. no. 12163); simple • Gamborg B-5 Basal Medium (Phytotechnology, cat. no. G398); simple • T4 DNA Ligase (Takara, cat. no. 2011A); simple • Bam HI-HF (NEB, cat. no. R3136S); simple • Stu I (NEB, cat. no. R0187S).

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: For overexpression studies, a pcDNA3.1 (−) construct containing the full murine Tspo cDNA ORF, a generous gift from Prof. V. Papadopoulos (McGill University) was transfected; the empty vector was used as a control in this instance. .. Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    Negative Control:

    Article Title: TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control
    Article Snippet: As a control for these studies a nonsilencing siRNA sequence (AllStars Negative Control, Qiagen, 1027281) was transfected. .. Experiments, unless otherwise indicated, were performed in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum or in the following saline solution (recording solution, RM): 125 mM NaCl (S5886), 5 mM KCl (P5405), 1 mM NaH2 PO4 (S5011), 20 mM HEPES (H3784), 5.5 mM glucose (G7021), 5 mM NaHCO3 (S5761), and 1 mM CaCl2 (C7902), (all from Sigma-Aldrich, UK) pH 7.4.

    Sample Prep:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: Paragraph title: Sample preparation for proteomic analysis ... Samples were diluted 1:5 in 25 mM Tris-HCl (Sigma-Aldrich, T1530), pH 8.2, and CaCl2 (Sigma-Aldrich, C3306) was added to a final concentration of 1 mM.

    Next-Generation Sequencing:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling. .. Heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich cat. no. F4135–500ML) Normal Goat Serum (NGS, Sigma-Aldrich cat. no. G6767–500ML) Triton X-100 (Sigma-Aldrich cat. no. T8787–100ML) Hoechst (Santa Cruz cat. no. 33258) Oligonucleotide pool (CustomArray) Primers (Sigma-Aldrich) dNTPs (New England Biolabs cat. no. N0447S) Agarose (Invitrogen cat. no. 16500–500) Sybr Safe (Invitrogen cat. no. S33102) Alexa488-conjugated goat anti-mouse (Life Sciences cat. no. ) Alexa594-conjugated goat anti-rabbit (Life Sciences cat. no. ) Mouse anti-FLAG (Sigma-Aldrich cat. no. F1804–50UG) Counterstain antibody not made in a mouse DG-52 mouse anti-SAG1 Alexa-488 labeling kit (Life Technology ) Midiprep kit (Macherey-Nagel cat. no. 740412.50) Miniprep kit (Zymo cat. no. D4054) DNeasy DNA extraction kit (Qiagen cat. no. 69504) QIAquick gel extraction kit (Qiagen cat. no. 28706) Gibson Assembly (New England Biolabs cat. no. E2611L) E. cloni 10G Supreme electrocompetent cells (VWR cat. no. 95024–012) AseI and associated NEBuffer 3.1 (New England Biolabs cat. no. R0526L) DNaseI (Sigma-Aldrich cat. no. DN25–100MG) Trypsin (Worthington cat. no. ) iProof and HF buffer (BioRad cat. no. 422840) Q5 DNA Polymerase (NEB cat. no. M0491L) BsaI and associated Buffer G (Thermo Scientific cat. no. ER0291) Calf Intestinal Phosphatase (CIP, New England Biolabs cat. no. M0290S) HFF cells (American Type Culture Collection cat. no. SCRC-1041) RH/Cas9 (American Type Culture Collection or by request) CAUTION Check for Cas9 expression and function before starting a screen by following steps 1–25 in the protocol.

    Concentration Assay:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: .. Samples were diluted 1:5 in 25 mM Tris-HCl (Sigma-Aldrich, T1530), pH 8.2, and CaCl2 (Sigma-Aldrich, C3306) was added to a final concentration of 1 mM. .. Proteins were digested by adding trypsin (Promega, V511A) 1:250 (enzyme:substrate) and incubated at 37°C overnight.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: On-slide tissue digestion for mass spectrometry based glycomic and proteomic profiling
    Article Snippet: Wear gloves and safety goggles while handling • Ammonium acetate(A7330-500 g, Sigma Aldrich), • Ammonium bicarbonate(A6141, Sigma Aldrich) • Tris-HCl(T3253, Sigma, pH 8), • Calcium Chloride(C5080, Sigma Aldrich), • Ammonium hydroxide, 25–30%(320145, Sigma Aldrich) CAUTION corrosive and an irritant by vapor or ingestion. .. Wear gloves and safety goggles while handling • Dithiothreitol (D0632, Sigma), • Iodoacetamide(163-2109, Biorad), • Trifluoro-acetic acid(A116-10X AMP, Fisher Scientific) • acetonitrile(A955-1, LC–MS grade, Fisher Scientific), • Water (W6-1, LC–MS grade, Fisher Scientific), • Formic acid(A117-10x-1AMP, LC–MS grade, Fisher Scientific) CAUTION flammable, corrosive.

    FACS:

    Article Title: AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells
    Article Snippet: .. For APC ANXA5/annexin V (ex/em maxima 650/660)-PI measurements, the cell pellets were resuspended in 50 µl FACS buffer (10 mM HEPES [AppliChem, 7365–45-9], 140 mM NaCl [Sigma-Aldrich, 31,434], 5 mM CaCl2 [Sigma-Aldrich, C3306], pH 7.4) with 0.8 µl APC ANXA5/annexin V and 0.8 µg/mL PI. .. Measurements were performed either with a FACS Accuri or FACS Canto II cytometer (BD Biosciences, Heidelberg, Germany).

    Hood:

    Article Title: CRISPR/Cas9-based genome-wide screening of Toxoplasma gondii
    Article Snippet: .. Na2 HPO4 (Sigma-Aldrich cat. no. 255793) NaCl (Sigma Aldrich cat. no. S7653) Glutathione (Sigma-Aldrich cat. no. G6013–5G) Adenosine Triphosphate (ATP, Sigma-Aldrich cat. no. A6419–1G) CaCl2 (Sigma-Aldrich cat. no. C5080–1KG) Chloramphenicol (Sigma-Aldrich cat. no. C3175–100MG) Pyrimethamine (Sigma-Aldrich cat. no. 46706–250MG) NaOH (Sigma-Aldrich cat. no. S2770) CAUTION Sodium hydroxide is a strong base that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling. .. HCl (Millipore cat. no. HX0603–4) CAUTION Hydrochloric acid is a strong acid that can cause severe skin burns and eye injuries; avoid contact with eyes, skin and clothing; use in a fume hood and wear protective eyewear and gloves during handling.

    Article Title: AAV-mediated gene targeting methods for human cells
    Article Snippet: BSA (Sigma, cat. no. A3912) Bromophenol blue (Sigma, cat. no. B-5525) CaCl2 ·2H2 O (Sigma, cat. no. C3306-500G) Potassium acetate (C2 H3 KO2 , Sigma, cat. no. P1190-500G) Calf intestinal alkaline phosphatase (CIP, New England Biolabs, cat. no. M0290S) DH10B electrocompetent cells (Invitrogen, cat. no. 18290015) Disodium EDTA·2H2 O (Sigma, cat. no. E5134-500G) Dispase (Invitrogen, cat. no. 17105-041) DMEM/F12 (Invitrogen, cat. no. 11320-033) DMEM high glucose (Invitrogen, cat. no. 10566-016) DMEM low glucose (Invitrogen, cat. no. 11885-084) DMSO (Sigma, cat. no. D4540) It is light sensitive. .. Handle inside a fume hood; wear protective clothing including gloves, plastic shield and safety glasses.

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  • 97
    Millipore atp measurement
    <t>AMD</t> RPE are more susceptible to oxidative stress and show lower mitochondrial activity. ( a and b ) Cell viability assays of AMD and control RPE treated with increasing concentrations of H 2 O 2 for 24 h ( a ) and 48 h ( b ). Higher susceptibility of the AMD RPE under oxidative stress conditions is observed in 48 h. ( c ) ROS production under stress is significantly higher in AMD RPE. ( d and e ) AMD RPE have significantly lower mitochondrial activity, as indicated by their <t>ATP</t> levels measured by a luminescence assay in the presence of hexokinase inhibitor. ( d ) ATP levels produced by mitochondria are significantly lower in AMD RPE as measured in the presence of hexokinase inhibitor. ( e ) ATP levels produced by glycolysis are higher in AMD RPE as measured in the absence of hexokinase inhibitor. The difference in ATP levels measured in the presence ( d ) and absence ( e ) of hexokinase inhibitor show glycolysis as the major source of ATP production in AMD RPE. Asterisks (*) indicate statistical significance, determined by the ANOVA analysis followed by Tukey's test ( P -value
    Atp Measurement, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AMD RPE are more susceptible to oxidative stress and show lower mitochondrial activity. ( a and b ) Cell viability assays of AMD and control RPE treated with increasing concentrations of H 2 O 2 for 24 h ( a ) and 48 h ( b ). Higher susceptibility of the AMD RPE under oxidative stress conditions is observed in 48 h. ( c ) ROS production under stress is significantly higher in AMD RPE. ( d and e ) AMD RPE have significantly lower mitochondrial activity, as indicated by their ATP levels measured by a luminescence assay in the presence of hexokinase inhibitor. ( d ) ATP levels produced by mitochondria are significantly lower in AMD RPE as measured in the presence of hexokinase inhibitor. ( e ) ATP levels produced by glycolysis are higher in AMD RPE as measured in the absence of hexokinase inhibitor. The difference in ATP levels measured in the presence ( d ) and absence ( e ) of hexokinase inhibitor show glycolysis as the major source of ATP production in AMD RPE. Asterisks (*) indicate statistical significance, determined by the ANOVA analysis followed by Tukey's test ( P -value

    Journal: Cell Death & Disease

    Article Title: Dysfunctional autophagy in RPE, a contributing factor in age-related macular degeneration

    doi: 10.1038/cddis.2016.453

    Figure Lengend Snippet: AMD RPE are more susceptible to oxidative stress and show lower mitochondrial activity. ( a and b ) Cell viability assays of AMD and control RPE treated with increasing concentrations of H 2 O 2 for 24 h ( a ) and 48 h ( b ). Higher susceptibility of the AMD RPE under oxidative stress conditions is observed in 48 h. ( c ) ROS production under stress is significantly higher in AMD RPE. ( d and e ) AMD RPE have significantly lower mitochondrial activity, as indicated by their ATP levels measured by a luminescence assay in the presence of hexokinase inhibitor. ( d ) ATP levels produced by mitochondria are significantly lower in AMD RPE as measured in the presence of hexokinase inhibitor. ( e ) ATP levels produced by glycolysis are higher in AMD RPE as measured in the absence of hexokinase inhibitor. The difference in ATP levels measured in the presence ( d ) and absence ( e ) of hexokinase inhibitor show glycolysis as the major source of ATP production in AMD RPE. Asterisks (*) indicate statistical significance, determined by the ANOVA analysis followed by Tukey's test ( P -value

    Article Snippet: To assay the mitochondrial activity in AMD and normal RPE, the ATP measurement was performed following 2-h incubation with or without 10 μ M of the bromopyruvate analog (3-BrPA), an inhibitor of the glycolytic enzyme hexokinase II (EMD Millipore, Billerica, MA, USA).

    Techniques: Activity Assay, Luminescence Assay, Produced

    Fluorescence anisotropy measurements (Δ r ) of equilibrium binding of ISWI to DNA and nucleosomes in the presence of nucleotides. (A) Equilibrium binding to a 20 bp FITC-labeled DNA substrate (25 nM) in the presence of ATP-γ-S. These data were analyzed using Scheme 1 as described in Experimental Procedures . The solid lines in the figure represent the fits of the data to this scheme, which returned the following values: 1/β A = 140 ± 30 μM, 1/β A,1 = 390 ± 70 μM, and 1/β 1,A = 42 ± 8 nM. (B) Equilibrium binding to a 20 bp FITC-labeled DNA substrate (25 nM) in the presence of ADP. The solid line in this figure represents the fit of equilibrium DNA binding data collected in the absence of nucleotide (Figure 1 A). (C) Equilibrium binding to an Alexa488-labeled 10N5 nucleosome substrate in the presence of ATP-γ-S. (D) Equilibrium binding to an Alexa488-labeled 10N5 nucleosome substrate in the presence of ADP. The solid lines in panels C and D are the fits of the equilibrium nucleosome binding data collected in the absence of nucleotides (Figure 1 C).

    Journal: Biochemistry

    Article Title: Quantitative Determination of Binding of ISWI to Nucleosomes and DNA Shows Allosteric Regulation of DNA Binding by Nucleotides

    doi: 10.1021/bi500224t

    Figure Lengend Snippet: Fluorescence anisotropy measurements (Δ r ) of equilibrium binding of ISWI to DNA and nucleosomes in the presence of nucleotides. (A) Equilibrium binding to a 20 bp FITC-labeled DNA substrate (25 nM) in the presence of ATP-γ-S. These data were analyzed using Scheme 1 as described in Experimental Procedures . The solid lines in the figure represent the fits of the data to this scheme, which returned the following values: 1/β A = 140 ± 30 μM, 1/β A,1 = 390 ± 70 μM, and 1/β 1,A = 42 ± 8 nM. (B) Equilibrium binding to a 20 bp FITC-labeled DNA substrate (25 nM) in the presence of ADP. The solid line in this figure represents the fit of equilibrium DNA binding data collected in the absence of nucleotide (Figure 1 A). (C) Equilibrium binding to an Alexa488-labeled 10N5 nucleosome substrate in the presence of ATP-γ-S. (D) Equilibrium binding to an Alexa488-labeled 10N5 nucleosome substrate in the presence of ADP. The solid lines in panels C and D are the fits of the equilibrium nucleosome binding data collected in the absence of nucleotides (Figure 1 C).

    Article Snippet: To separate ADP from ATP species, reaction mixtures were analyzed using thin liquid chromatography PEI-cellulose plates (EMD chemicals) in 0.6 M potassium phosphate (pH 3.4) buffer, quantified using a Typhoon Phosphor imager.

    Techniques: Fluorescence, Binding Assay, Labeling

    Identification of CK2 phosphorylation sites in mouse SIRT1. (A) Diagram of recombinant mouse SIRT1 fragments produced in E.coli. The a.a. number of the first and last a. a. for each fragment is as indicated. (B) SIRT1 fragments from (A) were incubated with CK2 α and 32 P[γ-ATP], subjected to SDS-PAGE, Coomassie staining and autoradiography. GST-PC, which contains the optimized CK2 phosphorylation site, was used as a positive control. (C) Evolutionarily conserved CK2 consensus sites located in the fragments phosphorylated by CK2 are marked (*). (D–H) Indicated Ser (S) residues in SIRT1 fragments (see Fig. 2A ) were mutated to Ala (A) individually or in combination. Fragments containing the mutation were incubated with CK2 α and 32 P[γ-ATP] and then subjected to SDS-PAGE, Coomassie staining and autoradiography. (I) CK2-dependent phosphorylation of full-length (FL) and S154/649/651/683A (4A) mutant SIRT1 in vitro . CK2 phosphorylation was performed and visualized as in (B).

    Journal: PLoS ONE

    Article Title: CK2 Is the Regulator of SIRT1 Substrate-Binding Affinity, Deacetylase Activity and Cellular Response to DNA-Damage

    doi: 10.1371/journal.pone.0006611

    Figure Lengend Snippet: Identification of CK2 phosphorylation sites in mouse SIRT1. (A) Diagram of recombinant mouse SIRT1 fragments produced in E.coli. The a.a. number of the first and last a. a. for each fragment is as indicated. (B) SIRT1 fragments from (A) were incubated with CK2 α and 32 P[γ-ATP], subjected to SDS-PAGE, Coomassie staining and autoradiography. GST-PC, which contains the optimized CK2 phosphorylation site, was used as a positive control. (C) Evolutionarily conserved CK2 consensus sites located in the fragments phosphorylated by CK2 are marked (*). (D–H) Indicated Ser (S) residues in SIRT1 fragments (see Fig. 2A ) were mutated to Ala (A) individually or in combination. Fragments containing the mutation were incubated with CK2 α and 32 P[γ-ATP] and then subjected to SDS-PAGE, Coomassie staining and autoradiography. (I) CK2-dependent phosphorylation of full-length (FL) and S154/649/651/683A (4A) mutant SIRT1 in vitro . CK2 phosphorylation was performed and visualized as in (B).

    Article Snippet: In vitro kinase assay To identify CK2 phosphorylation sites, recombinant full-length or truncated His-tagged or GST-tagged SIRT1 proteins were incubated in kinase buffer (20 mM HEPES at pH 7.0, 10 mM MgCl2 , 1 mM DTT) containing 100 µM cold ATP, 1 µCi (3000 mCi/mmole) 32 P[γ-ATP] and 20 ng CK2α enzyme (Millipore) for 20 min at 30°C.

    Techniques: Recombinant, Produced, Incubation, SDS Page, Staining, Autoradiography, Positive Control, Mutagenesis, In Vitro

    ADP/ATP exchange assays with human ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis. ( A for description). ( B ) Assay validation with known compounds (10 μM) in isolated yeast mitochondria (left panel) and L. lactis (right panel) using the hANT2 isoform. The ADP/ATP exchange activity was expressed as the percent relative to the DMSO control. Mean ± SD from a single experiment. ANT, adenine nucleotide translocase; TTFA, thenoyltrifluoroacetone; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; MPTP, mitochondrial permeability transition pore

    Journal: Journal of biomolecular screening

    Article Title: Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast

    doi: 10.1177/1087057115624637

    Figure Lengend Snippet: ADP/ATP exchange assays with human ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis. ( A for description). ( B ) Assay validation with known compounds (10 μM) in isolated yeast mitochondria (left panel) and L. lactis (right panel) using the hANT2 isoform. The ADP/ATP exchange activity was expressed as the percent relative to the DMSO control. Mean ± SD from a single experiment. ANT, adenine nucleotide translocase; TTFA, thenoyltrifluoroacetone; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; MPTP, mitochondrial permeability transition pore

    Article Snippet: The ADP/ATP exchange assay was carried out in a filter plate format using a 96-well MultiScreenHTS FC filter plate (Millipore, Billerica, MA).

    Techniques: Isolation, Activity Assay, Permeability