A549 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [0.15 mol/L sodium chloride, 1% Triton‐X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 5 mmol/L ethylene diamine tetraacetic acid (EDTA), 50 mmol/L Tris (pH 8)] (Sigma, EDTA from GE Healthcare, Buckinghamshire, UK, Tris from Merck Millipore, Darmstadt, Germany), supplemented with complete protease inhibitor (Roche, Mannheim, Germany). Protein concentrations were measured using the Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 15 µg protein was separated on NuPage Mini 3‐8% Tris‐Acetate gels (Invitrogen, Waltham, Massachusetts, USA) and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). For probing of ABCA3‐HA, rat anti‐HA monoclonal antibody (Roche) and rabbit anti‐rat IgG (H + L) HRP secondary antibody (Southern Biotechs, Birmingham, AL) were used. β‐Actin (Santa Cruz, Dallas, TX) probing served as a loading control. SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was used for detection. Densitometric analysis was performed with Image J software.
Ethylene diamine tetraacetic acid (edta)
Manufactured by GE Healthcare
Sourced in United States, Germany
EDTA is a chemical compound commonly used as an anticoagulant in laboratory equipment and procedures. It functions by binding to metal ions, preventing them from participating in enzymatic reactions that lead to blood clotting.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by GE Healthcare (7 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Biacore x100, by Cytiva (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hepes, by GE Healthcare (2 mentions)
Casy ton buffer solution, by Roche (2 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Anti ha rat monoclonal antibody, by Roche (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
X100 2, by Cytiva (1 mentions)
Tween 20, by Carl Roth (1 mentions)
Up50h ultrasonic processor, by Hielscher (1 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Bradford kit, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by GE Healthcare (1 mentions)
Abx pentra 400, by Horiba (1 mentions)
Penicillin streptavidin, by Thermo Fisher Scientific (1 mentions)
21 protocols using ethylene diamine tetraacetic acid (edta)
1
ABCA3 Protein Expression Analysis
2
Quantifying Cell Adhesion via LDH
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Cell adhesion in the presence of Mg2+ (total) and EDTA (non-specific) was assessed calorimetrically through the measurement of lactate dehydrogenase (LDH) activity release from adhered cells into the media.
All cell lines were maintained in a humidified incubator with 5 % CO2 at 37 °C in DMEM containing 10 % fetal bovine serum and 1 % streptavidin/penicillin. Prior to cell adhesion experiments, cells were detached from the cell culture flasks with 0.05 % trypsin/0.02 % EDTA (GE Healthcare), washed and re-suspended in serum free DMEM.
All cell lines were maintained in a humidified incubator with 5 % CO2 at 37 °C in DMEM containing 10 % fetal bovine serum and 1 % streptavidin/penicillin. Prior to cell adhesion experiments, cells were detached from the cell culture flasks with 0.05 % trypsin/0.02 % EDTA (GE Healthcare), washed and re-suspended in serum free DMEM.
3
Biacore Assay for AH/AvFc Binding Affinity
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The binding affinity (KD) of AH or AvFc to gp120 was measured on a Biacore X100 2.0 instrument at ambient temperature. Recombinant His-tagged gp120 (Q769.h5, Immune Technology, #IT-001-0012p; SF162, Immune Technology, #IT-001-0028p-PBS; and ZM53M.PB12 Immune Technology, #IT-001-RC8p) proteins were captured on a sensor chip NTA following the manufacturer’s instructions to a surface density of about 30 RUs. A reference flow cell was utilized to correct response contributions such as bulk shifts that occur equally in the sample and reference flow cells. Serial dilutions of AH or AvFc were made in running buffer (HPS-P+ with 50 μM EDTA, GE Healthcare) and injected at a flow rate of 5 μL/min for a contact time of 120 s and a dissociation time of 1,200 s. A blank cycle (running buffer) was performed, and all sample injections were blank subtracted to correct the sensorgrams for drifts and other disturbances that affect the reference subtracted curve. Between sample injections, the system was washed with NTA wash buffer, with the surface regenerated with the NTA regeneration solution. A replicate of a non-zero concentration of AH or AvFc and the blank were injected in each experiment for double referencing, thus verifying the reliability of the immobilized chip throughout the experiment. The data were assessed by steady-state binding analysis.
4
Metabolite and Enzyme Analysis in Mucosa
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For the analyses of metabolites and enzyme activities, 30 mg of powdered mucosa tissue was homogenized in 300 µl lysis puffer containing 10 mM HEPES (Thermo Fisher Scientific, Schwerte, Germany), 1% (v/v) Tween20 (Carl Roth, Karlsruhe, Germany), 1 mM EDTA (GE Healthcare, Munich, Germany), 10 mM NaF (Thermo Fisher Scientific), 0.1% (v/v) Triton X-100 (GE Healthcare), 0.5% (v/v) DOC (Sigma-Aldrich), 0.1% (w/v) SDS (USB Corporation, Cleveland, OH, USA) with 0.5 cycles and 80% amplitude (20-times) Ultrasonic Processor UP50H (Hielscher Ultrasound Technology, Teltow, Germany)54 (link). The homogenized extract was centrifuged at 3000×g for 20 min at 4 °C. The supernatant was used to measure glucose and lactate concentrations and aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) activities photometrically (Abx Pentra 400; Horiba, Kyoto, Japan) using kits for glucose (no. A11A01667, Axon Lab, Reichenbach, Germany), lactate (no. A11A01721, Axon Lab), AST activity (no. A11A01629, Axon Lab) and GLDH activity (LT-GD 0010, Labor + Technik Eberhard Lehmann GmbH, Berlin, Germany). Protein concentrations of the extracts were measured using the Bradford kit (Thermo Fisher Scientific). Metabolites and enzyme activities were normalized to the protein concentration of the mucosa extract.
5
Adhesion and Spreading of HT1080 Cells and Platelets
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HT1080 cells derived from a human fibrosarcoma were obtained from the European Collection of Animal Cell Cultures, Porton Down, UK. HT1080s were maintained in a humidified incubator with 5 % CO2 at 37 °C in DMEM (Gibco), containing 10 % fetal bovine serum (Gibco) and 1 % streptavidin/penicillin (Life Technologies). Prior to cell adhesion/spreading experiments, HT1080 s were detached from the cell culture flasks with 0.05 % trypsin/0.02 % EDTA (GE Healthcare), washed and re-suspended in serum free DMEM. Platelets were obtained from human platelet rich plasma provided by the National Health Service Blood and Transplants authority in accordance with the Declaration of Helsinki. Platelets were prepared by centrifugation for 15 min, 240 g, the pellet discarded and 1 μl of Prostaglandin E1 (100 μg/ml in ethanol) added per ml of platelet supernatant. The platelet suspension was centrifuged at 640 g for 10 min and the platelet pellet resuspended in tyrodes buffer (140 mM NaCl, 5.6 mM Glucose, 2 mM MgCl2, 0.4 mM NaH2PO4, 12 mM NaHCO3, 2.7 mM KCl, 10 mM HEPES pH 7.4) to a density of 1 × 108/ml.
6
Annexin A1 Regulation in Cancer
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Ethical approval for this investigation was obtained from the Research Ethics Committee, the University of south China of Medicine. Participants had provided their written informed consent to participate in this study, and the ethics committees had approved this consent procedure.GV146-ANXA1 and GV-102-ANXA1-RNAi plasmids and Lipofectamine 2000 were purchased from GeneChem Co., Ltd. (Shanghai, China) and Invitrogen Life Technologies, respectively. Transwell chamber and Matrigel were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Bromophenol blue, EDTA, DMSO, Coomassie Brilliant Blue R-250, molecular weight marker, Tris-base, SDS, glycine, TFA, second antibodies-conjugated with horseradish peroxidase, PVDF membrane, and Protein G-Sepharose beads were purchased from GE Healthcare Life Sciences, USA. Mouse monoclonal anti-ANXA1, anti-Vimentin antibody, and IgG-antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal anti-S100A9 antibody was purchased from Abcam Biotechnology. Mercaptoethanol, iodoacetamide, and HCl were purchased from Sigma–Aldrich (St. Louis, MO, USA). All buffers were prepared using Milli-Q water.
7
SPR Analysis of ECRG4-2xCTLD-Fc Interaction
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The binding parameters of 2 × CTLD–Fc to ECRG4 were determined with the ProteOn™ XPR36 Protein Interaction Array System (Bio-Rad). His-tagged ECRG4 peptides were immobilised indirectly with an anti-His-tag antibody. Briefly, an anti-His × 8 antibody (Qiagen) was diluted in 10 mM acetate buffer (pH 4.5) and immobilised on an ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS)-preactivated ProteOn™ GLM Sensor Chip (Bio-Rad) with amine coupling. The unreacted carboxyl groups on the chip were blocked by the injection of 1 M ethanolamine HCl. The His-tagged Ecrg4 peptides were diluted to 10 μM in HEPES-buffered saline (HBS; 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20; GE Healthcare) and injected for 300 s onto the anti-His-antibody-coated chip for capture. HBS was used as the running buffer in all SPR experiments. For determination of the kinetic parameters of the interaction, different concentrations (250 nM, 125 nM, 62.5 nM, 31.3 nM, 15.6 nM) of the 2 × CTLD–Fc fusion were injected. The injection time was 60 s, and the dissociation time was 120 s. The data were processed with ProteOn™ Manager software. All kinetic parameters, such as the dissociation rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (Kd), were calculated with the Langmuir model.
8
Isolation of Human Splenocytes
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We obtained human spleen tissues from donors with ruptured spleen caused by trauma, with informed consent from the donors. Studies were approved by the Human Research Ethics Committee of Xuzhou Mine Hospital with the protocol #XZKSYY2021-002.
Human splenocytes were isolated from spleen tissue as reported (Mittag et al., 2011 (link)). Briefly, spleen tissue was disrupted with ophthalmic scissors and then digested with collagenase (2 mg/mL; Worthington Biochemical, Lakewood, NJ, USA) in the presence of DNase (0.1 mg/mL; Roche, Mannheim, Germany) at 5g tissue/20mL RPMI (Roswell Park Memorial Institute) 1640 medium (Invitrogen; Carlsbad, CA, USA) for 20 min at room temperature, followed by addition of 5mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 5 min at room temperature. Undigested fragments were removed by filtering through a 70-mm sieve. After washing in PBS with 1% FBS and 1mM EDTA, spleen mononuclear cells were purified by Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density centrifugation.
Human splenocytes were isolated from spleen tissue as reported (Mittag et al., 2011 (link)). Briefly, spleen tissue was disrupted with ophthalmic scissors and then digested with collagenase (2 mg/mL; Worthington Biochemical, Lakewood, NJ, USA) in the presence of DNase (0.1 mg/mL; Roche, Mannheim, Germany) at 5g tissue/20mL RPMI (Roswell Park Memorial Institute) 1640 medium (Invitrogen; Carlsbad, CA, USA) for 20 min at room temperature, followed by addition of 5mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 5 min at room temperature. Undigested fragments were removed by filtering through a 70-mm sieve. After washing in PBS with 1% FBS and 1mM EDTA, spleen mononuclear cells were purified by Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density centrifugation.
9
Cell Culture Protocols for Fibrosarcoma and Glioma
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Human fibrosarcoma cells, HT1080, were obtained from the European Collection of Animal Cell Cultures (Porton Down, UK). Rugli cells, derived from a rat glioma, were a kind gift from Dr. J. Gavrilovic, University of East Anglia, UK. Both cell lines were maintained at 37 °C in Dulbecco's modified Eagle's medium (DMEM, Sigma–Aldrich) containing 10% (v/v) foetal bovine serum (Sigma–Aldrich) and 1% (v/v) PenStrep (Life Technologies). Prior to cell binding or cell spreading experiments, cells were detached from cell culture flasks at a confluence of about 75% with 0.05% trypsin/0.02% EDTA (GE Healthcare), washed and resuspended in serum free DMEM.
10
Antibody-Based Protease Profiling
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Antibodies against complement components C1s (Sheep Anti‐human C1s, AF2060, and mouse anti‐human C1s MAB2060) and C1r antibodies (AF1807‐SP) and antibodies recognizing human MMP‐9 (AF911) and human MMP‐2 (AF902) were purchased from R&D Systems (Minneapolis, MN, USA). HRP‐conjugated antibodies were purchased from Jackson Human Research (Cambridgeshire, UK). Recombinant C1s (H00000716‐P01) was from Abnova (Taipei City, Taiwan). Gelatin from bovine skin, type B (G9391) for zymography analysis was from Sigma (Sigma‐Aldrich, St Louis, MO, USA). EDTA, protein G‐Sepharose 4 Fast Flow (71‐7083‐00) and gelatin‐Sepharose 4B (17‐0956‐01) were purchased from GE Healthcare (Chicago, IL, USA), Coomassie brilliant blue (B0149) was from Sigma. EDTA (VWR‐ 20302.260) was from BDH‐chemicals (Brooklyn, NY, USA).
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