Cell fusion process can be tuned by changing cell-cell interactions via EGTA treatment, which is a calcium chelator that can disrupt cadherin functions. For EGTA treatment, a stock solution of EGTA (10 mM, Sigma) was prepared by dissolving EGTA in water and adjusting pH with NaOH and HCl. The stock solution was diluted in culture media to different concentrations (control, 0.1 μM, and 0.15 μM), and cells were treated 1 hr after cell seeding. The number of MGCs were calculated after 4, 14, 26, and 47 hr treatment, respectively. In addition, the cell fusion can also be affected by tuning contractile forces of cells. ROCK Inhibitor Y27632 can reduce the contractile forces, while lysophosphatidic acid (LPA) can activate ROCK signals and increase contractile forces. For Y27632 (Cayman Chemical, Ann Arbor, MI) treatment, a stock solution (10 mM in DMSO) was prepared and diluted in culture media to 10 μM. Cells were treated 1 hr after cell seeding. For LPA (Invitrogen) treatment, a stock solution (10 mM in DMSO) was prepared and diluted in culture media to 10 μM. Cells were treated 1 hr after cell seeding.
Ethylene glycol tetraacetic acid (egta)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, Macao, Sao Tome and Principe, France, China, Japan, Poland, Hungary, Canada, Ireland, Spain
EGTA is a chemical compound commonly used as a chelating agent in laboratory settings. It is capable of binding to metal ions, such as calcium, and is often utilized in experimental procedures to control the concentration of specific ions within a sample.
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Lab products found in correlation
Hepes, by Merck Group (98 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (82 mentions)
Sodium chloride (nacl), by Merck Group (66 mentions)
Mgcl2, by Merck Group (54 mentions)
Potassium chloride (kcl), by Merck Group (49 mentions)
Bovine serum albumin (bsa), by Merck Group (47 mentions)
Sucrose, by Merck Group (43 mentions)
Triton x 100, by Merck Group (36 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (36 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (33 mentions)
Cacl2, by Merck Group (31 mentions)
Mannitol, by Merck Group (30 mentions)
Dithiothreitol (dtt), by Merck Group (30 mentions)
Pipes, by Merck Group (26 mentions)
Thapsigargin, by Merck Group (24 mentions)
Adenosine triphosphate (atp), by Merck Group (22 mentions)
Rotenone, by Merck Group (21 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (21 mentions)
Protease inhibitor cocktail, by Merck Group (19 mentions)
Leupeptin, by Merck Group (19 mentions)
646 protocols using ethylene glycol tetraacetic acid (egta)
1
Tuning Cell Fusion via Cell-Cell Interactions
2
Quantifying Cellular Oxidative Stress
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Total O2 •-levels were determined using the cytochrome C reduction assay [48] . Briefly, cell pellets were sonicated in cold lysis buffer containing HEPES buffer (20 mM, pH 7.2, Calbiochem), ethylene glycol tetraacetic acid (1 mM, Sigma), mannitol (210 mM, Sigma) and sucrose (70 mM, Sigma). Equal amounts of homogenate (100 µg) were incubated with cytochrome C (50 µM, Sigma) at 37°C for 60 minutes before absorbances were measured at 550 nm.
NADPH oxidase activity was measured with the lucigenin chemiluminescence assay [51] .
Briefly, samples of homogenates (~100 µg) were processed as above then incubated at 37°C in assay buffer containing potassium phosphate buffer (50 mM, pH 7.0, Sigma), ethylene glycol tetraacetic acid (1 mM), sucrose (150 mM) and lucigenin (5 µM, Sigma). The assay buffer also contained the specific inhibitors for other ROS-generating enzymes; nitric oxide synthase (NG-nitro-L-arginine methyl ester, 100 µM, Sigma), mitochondrial complex I (rotenone, 50 µM, Sigma), xanthine oxidase (allopurinol, 100 µM, Sigma) and cyclooxygenase (indomethacin, 50 µM, Sigma). After 15 minutes, NADPH (100 µM; Calbiochem) was added to initiate the reaction. The reaction was monitored every minute for 2 hours and the rate of reaction calculated. Buffer blanks were also run for both assays and subtracted from the data.
NADPH oxidase activity was measured with the lucigenin chemiluminescence assay [51] .
Briefly, samples of homogenates (~100 µg) were processed as above then incubated at 37°C in assay buffer containing potassium phosphate buffer (50 mM, pH 7.0, Sigma), ethylene glycol tetraacetic acid (1 mM), sucrose (150 mM) and lucigenin (5 µM, Sigma). The assay buffer also contained the specific inhibitors for other ROS-generating enzymes; nitric oxide synthase (NG-nitro-L-arginine methyl ester, 100 µM, Sigma), mitochondrial complex I (rotenone, 50 µM, Sigma), xanthine oxidase (allopurinol, 100 µM, Sigma) and cyclooxygenase (indomethacin, 50 µM, Sigma). After 15 minutes, NADPH (100 µM; Calbiochem) was added to initiate the reaction. The reaction was monitored every minute for 2 hours and the rate of reaction calculated. Buffer blanks were also run for both assays and subtracted from the data.
3
Hepatocyte Differentiation and Infection
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Prior to inoculation, differentiated HepaRG cells were treated with 1X EGTA (Sigma Aldrich) for 1h at 37°C, 5% CO2 and were inoculated with 500 ge per cell in complete medium containing, 1.8% DMSO, 4% PEG 8000, 10-fold the normal amount of hemisuccinate hydrocortisone and 1X EGTA. Cells were incubated 1h at room temperature and 20h at 37°C and 5% CO2. Cells were washed five times in complete medium containing 1.8% DMSO. PHH were inoculated using the same procedure in the absence of EGTA treatment. NAPs were resuspended in PBS and added to cells medium at the indicated time and concentrations.
4
Nuclear Fractionation and Co-Immunoprecipitation
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Regarding IP experiments, nuclear cell extracts of HeLa cells were obtained by a sequential lysis with buffer A [10 mM HEPES pH 7.9 (Fisher scientific, BP299-100), 10 mM KCl (Sigma-Aldrich, 44,675), 0.1 mM EDTA (Sigma-Aldrich, E9884), 0.1 mM EGTA (Millipore, 324,628), 0.6% NP-40 (Millipore, 492,016), 1 mM DTT (Roche, 10,197,777,001) and 1 mM PMSF (Roche, P7626)] and buffer C [20 mM HEPES, pH 7.9, 400 mM NaCl (Sigma-Aldrich, S9888), 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF], which resulted respectively in cytosolic and nuclear fraction isolation. Protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23,225) and 700 μ g of nuclear fraction were immunoprecipitated by using 4 µg of the indicated antibody and 50 µl of Dynabeads Protein A/G (Invitrogen, 10002D/10004D), according to manufacturer’s instructions. The following primary antibodies were used for IP: mouse mAb anti-TERF2/TRF2 4A794 (Millipore, 05–521), rabbit pAb anti-HMGB1 (Abcam, Ab18256). IP with anti-normal mouse IgG (Santa Cruz Biotechnology, sc-2025) and anti-normal rabbit IgG (Santa Cruz Biotechnology, sc-3888) were used as respective negative controls.
5
Isolation of Embryonic Germ Cell Progenitors
6
Isolation of Embryonic Germ Cell Progenitors
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PGC isolation was carried out as previously described4 (link). Briefly, the embryonic trunk (E10.5) or genital ridge (E11.5-E14.5) was digested at 37°C for 3 min using 0.05% Trypsin-EDTA (1x) (Gibco) or TrypLE Express (Thermo). Enzymatic digestion was followed by neutralization with DMEM/F-12 (Gibco) containing 15% foetal bovine serum (Gibco) and manual dissociation by pipetting. Following centrifugation, cells were re-suspended in DMEM/F-12 supplemented with hyaluronidase (300 µg/ml; Sigma), and a single cell suspension was generated by manual pipetting. Following centrifugation, cells were re-suspended in ice-cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml, Sigma). GFP positive cells were isolated using an Aria IIu (BD Bioscience) or Aria III (BD Bioscience) flow cytometer and sorted into ice cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml, Sigma).
7
Isolation and Purification of Mitochondria
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The brain, heart, muscle, liver, testis, and MEF mitochondria were isolated in MS-EGTA buffer [225 mM mannitol, 75 mM sucrose, 5 mM Hepes, and 1 mM EGTA (pH 7.4); Sigma-Aldrich]. The various tissues were minced into 2 mm by 2 mm pieces and then homogenized using a glass and Teflon tissue homogenizer (8 to 15 strokes, depending on the tissue) on ice and in MS-EGTA buffer. Tissue homogenates were then subjected to a 2500g centrifugation for 5 min (1×), and the supernatants were then centrifuged at 11,500g for 10 min. The MEFs were grown to confluency on four Nunc Bioassay Dishes (Sigma-Aldrich) and then harvested and pelleted in MS-EGTA buffer. The cells were then suspended in 7 ml of buffer and homogenized with a glass and Teflon tissue homogenizer (10 strokes). MEF homogenates were then subjected to a 2500g centrifugation for 5 min (2×), and the supernatants were then centrifuged at 11,500g for 10 min. In both situations, pellets were then washed and centrifuged 2× (11,500g) and then snap-frozen or suspended in KCl buffer [125 mM KCl, 20 mM Hepes, 2 mM MgCl2, 2 mM KH2PO4, and 40 μM EGTA (pH 7.2); Sigma-Aldrich].
8
Isolation of Cardiac Mitochondrial Subfractions
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The entire heart was used for mitochondrial isolation using a previously described protocol [26 (link)]. Hearts were minced for 5 min in 10× mg-volume stable buffer [solution 1: 100 mM KCl (Sigma, St. Louis, MO, USA, P4504), 50 mM MOPS (Sigma M1254), 5 mM MgSO4 (Sigma M2643), 1 mM EGTA (Sigma E4378), 1 mM ATP (Sigma M2643), 0.2% BSA (Sigma A6003), pH 7.4 at 4 °C]. Contents were briefly processed with a mechanical homogenizer, followed by a PFTE Dounce homogenizer and brief centrifugation (500× g, 10 min, 4 °C) to separate the SS (supernatant) from IMF (pellet) mitochondria. The IMF subfraction was processed through mechanical homogenization again and underwent trypsin digestion [1× mg-volume (5 mg trypsin/g of tissue; Sigma T1426) prepared in solution 1]. Digestion was terminated with the addition of 10× mg-volume of solution 1. SS and IMF mitochondria underwent several washing steps with solution 1 and centrifugation (3500× g, 10 min, 4 °C), with a final pelleting spin in solution 2 (solution 1 without BSA). The mitochondrial pellet was resuspended in 250 µL of resuspension buffer [220 mM mannitol (Sigma M9467), 70 mM Sucrose (Sigma S9378), 10 mM Tris-HCl (VWR 0278, Radnor, PA, USA) and 1 mM EGTA (Sigma E4378), pH 7.4 at 4 °C], using separate PFTE Dounce homogenizers for each fraction.
9
Visualization of Plant Root Cell Nuclei
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For 6-day-old seedlings, sections (5 mm) of the root tip were fixed in a 4% solution of paraformaldehyde (Sigma Aldrich, Saint Louis, MO, USA) in PHEM buffer, pH = 6.9 (60 mM PIPES (Sigma Aldrich, USA), 25 mM HEPES (Sigma Aldrich, USA), 10 mM EGTA (Sigma Aldrich, USA), and 2 mM MgCl2 (Sigma Aldrich, USA) for 1.5 h at room temperature. The fixative was washed with PHEM buffer. The samples were incubated 7 min in 0.4 M mannitol containing 4% cellulase (Sigma Aldrich, USA) and 5 mM EGTA, then washed with PBS buffer; after incubation in the enzyme, the roots were transferred to coverslips and separated into individual cells. The prepared preparations were dried in the refrigerator at +4 °C for 24 h and placed in Mowiol U-44 (Hoechst, Germany) with the addition of DAPI (1 μL/1 mL) (4,6-diamidino-2-phenylindole) (Sigma Aldrich, USA). The samples were analyzed under an Olympus BX51 fluorescence microscope (Japan), ×100 objective. Images were obtained using Color View digital camera (Germany). ImageJ software was used to measure fluorescence intensity.
10
Epithelial Barrier Permeability Assay
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1 × 105 Eph4 cells were seeded onto the transwell inserts (cellulose mixed ester, 24-well, pore size 0.4 μm, translucent, BD Falcon), and cultured for four days. After TER values had reached a plateau, media were changed from D-MEM high glucose to Opti-MEM (Thermo Fisher Scientific), and 50 μg of mouse IgG or the mAb 67-2 were added to the lower chamber. 2 mM EGTA (Sigma) was applied to the upper chamber. TER value was measured with cellZscope instrument (CellSeed, Tokyo, Japan).
For the paracellular tracer flux assay, 10 min after applying mouse IgG, 67-2 or EGTA, 1 mg/ml fluorescein isothiocyanate (FITC)-labeled dextran with the molecular mass of 3-4 kDa, 70 kDa and 250 kDa (Sigma) was added to the upper chamber. After incubation for 2 h, the concentration of each tracer in the lower chamber was determined by measuring fluorescence with microplate reader (Varioskan, Thermo Fisher Scientific).
For the paracellular tracer flux assay, 10 min after applying mouse IgG, 67-2 or EGTA, 1 mg/ml fluorescein isothiocyanate (FITC)-labeled dextran with the molecular mass of 3-4 kDa, 70 kDa and 250 kDa (Sigma) was added to the upper chamber. After incubation for 2 h, the concentration of each tracer in the lower chamber was determined by measuring fluorescence with microplate reader (Varioskan, Thermo Fisher Scientific).
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