Adipose tissue derived mesenchymal stem cells from 4 horses (mixed breeds and sexes, aged 15 ± 4.64 years) and 3 dogs (mixed breeds and sexes, aged 4.81 ± 3.51 years) were cultured to obtain nanoparticles as previously described. After obtaining 24 ml per donor of cell culture supernatant containing EVs, as described above, we divided it into 5 ml reaction tubes (Sarstedt, Germany), so that a total of 6 tubes with a volume of 4 ml from each donor were available for exposure to different storage conditions. These tubes were processed as follows: EDTA (Carl Roth, Germany) was added to obtain a final concentration of 5 mM (tube 1), TRE (Carl Roth, Germany) was added to three reaction tubes to obtain a final concentration of 25 µM (tube 2), 50 µM (tube 3) or 250 µM (tube 4), one tube as control of fresh (tube 5) and one tube as frozen samples (tube 6) without addition of TRE or EDTA. The size and concentration of EVs in the fresh reaction tubes (tube 5) were measured immediately after EVs purification by nanoparticle tracking analysis (NTA), whereas all other preparations were frozen at − 20 °C for 8 days (tubes 1–4, 6). After the freezing period, the reaction tubes were thawed in a 37 °C water bath and subsequently measured by NTA.
Ethylene diamine tetraacetic acid (edta)
Manufactured by Carl Roth
Sourced in Germany, Switzerland, United States
EDTA is a chemical compound commonly used as a chelating agent in laboratory applications. It is effective at forming stable complexes with various metal ions, including calcium, magnesium, and heavy metals. EDTA is widely utilized in analytical procedures, sample preparation, and as a preservative in some laboratory reagents.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Dispase, by Roche (4 mentions)
Sodium chloride (nacl), by Avantor (4 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Anti apc microbeads, by Miltenyi Biotec (3 mentions)
Collagenase p, by Roche (3 mentions)
Anti cd45 nanobeads, by BioLegend (3 mentions)
Mgcl2, by Merck Group (3 mentions)
Hepes, by Carl Roth (3 mentions)
Bovine serum albumin (bsa), by Carl Roth (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Bio-Rad (3 mentions)
N laurylsarcosine, by Carl Roth (3 mentions)
Peqgold agarose, by Avantor (3 mentions)
Genepath electrophoresis gel cell, by Bio-Rad (3 mentions)
Proteinase k, by Carl Roth (3 mentions)
67 protocols using ethylene diamine tetraacetic acid (edta)
1
Storage Conditions for Equine and Canine EVs
2
Oxytocin Plasma Levels Protocol
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After decapitation, 500 μl blood was collected and 10 μl EDTA solution (0.8 mg/ml EDTA, Carl Roth, Karlsruhe) was added. Next, 7.5 μl of aprotinin solution (10 mg/ml aprotinin, Sigma-Aldrich, St. Louis) was added to 500 μl of EDTA blood and centrifuged at 4°C for 5 min at 1600 × g. The supernatant was stored at -80°C until measurement. Oxt plasma levels were determined via radioimmunoassay (RIA, RIAgnosis, Sinzing, Germany).
3
Skin Immune Cell Isolation Protocol
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Four hours after the injection of L3 larvae and/or agonists, the skin (approx. 1 cm2) was excised post mortem. Half of the skin was stored in 700 µL Trizol (QIAGEN, Hilden, Germany) at −20 °C until RNA isolation. The other half was minced and incubated at 37 °C on a shaker (350 rpm) for 75 min in an RPMI medium (Life technologies Corporation, Grand Island, NY, USA), supplemented with 10% FCS (PAN Biotech, Aidenbach, Germany), 1% Penicillin (10,000 units/mL)/Streptomycin (10 mg/mL) (Life technologies Corporation, Grand Island, NY, USA), 2 mM L-Glutamine (Life technologies Corporation, Grand Island, NY USA), 0.25 mg/mL Liberase TL (Hoffmann-La Roche Ltd., Basel, Switzerland), and 0.5 mg/mL DNase I (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was stopped with RPMI medium supplemented with 10 mM EDTA (Carl Roth, Karlsruhe, Germany) and 2% FCS. Cells were passed through a 70 µm cell strainer (Miltenyi Biotec, Bergisch Gladbach, Germany), centrifuged at 400× g for 5 min at 4 °C and taken up in MACS buffer (PBS, 1% FCS (PAN Biotech, Aidenbach, Germany), 2 mM EDTA (Carl Roth, Karlsruhe, Germany)). Cells were then counted with a CasyR TT Cell Counter + Analyser System (Schärfe Systems, Reutlingen, Germany), and 1 × 106 cells were analyzed by flow cytometry.
4
Amniotic Membrane Decellularization Protocol
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Excess blood was removed by washing with Hank’s balanced salt solution (HBSS; Life Technologies, Carlsbad, CA, USA. The amniotic membrane (AM) was mechanically peeled from the underlying chorion and washed several times in HBSS to remove blood. Subsequently, AM was washed with phosphate buffered saline without calcium and magnesium (PBS−/−, Life Technologies) and placed in lysis buffer (10 mM Tris and 0.1% EDTA (both Carl-Roth, Karlsruhe, Germany) for 1 h at room temperature, followed by incubation in 0.5% sodium dodecyl sulfate (SDS; Carl-Roth) solution for 4 h at room temperature with constant stirring. Next, the AM was washed in PBS three times at room temperature and finally overnight at 4 °C under constant agitation.
Decellularized AM (DeAM) was cut into appropriate pieces and lyophilized.
Decellularized AM (DeAM) was cut into appropriate pieces and lyophilized.
5
CatSper-Activity-Index for Sperm Motility
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The CatSper-Activity-Test was performed on men undergoing semen analysis in the course of a fertility workup, provided that the ejaculate featured a total sperm count of at least 5 × 106 and a minimum of 10% total motile sperm. We also performed the test on ejaculates from donors. A 20-μL aliquot of the ejaculate was diluted tenfold in HTF+ (Buffer A) and HTF0Ca containing 10 μM progesterone (Buffer B) or Buffer B including 5 mM EDTA (Carl Roth). To derive the CatSper-Activity-Index (CAI), the fraction of motile sperm in Buffer A and Buffer B was determined after 15 or 30 minutes and in Buffer B including EDTA after 30 or 60 minutes according to the equation:
6
Cultivation of HeLa cells with low connexin
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HeLa cells with a low endogenic connexin expression were a gift of K. Willecke. They were cultivated in an appropriate cell culture flask with culture medium consisting of Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (Sigma-Aldrich, Darmstadt, Germany), 100 U/mL penicillin G, and 100 µg/mL streptomycin (Thermo Fisher Scientific). Medium for transfected cells was additionally supplemented with 800 µg/mL G418 (Merck KGaA, Darmstadt, Germany). All steps were performed under sterile conditions (laminar air flow hood). The cultures were incubated at 37 °C in a humidified atmosphere with 5% CO2 in an incubator. Cells were regularly passaged after they had reached a confluence of 80–90%. The old medium was taken off and the cells were washed twice with sterile PBS to remove residual medium. The cells were detached using a trypsin 0.01% (Lonza Group Ltd., Basel, Switzerland) solution containing 0.6 mM EDTA (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) with incubation at 37 °C for 2–3 min.
7
Isolation of Intestinal Epithelial and Lamina Propria Cells
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After dissection, colons were excised and flashed with RPMI medium supplemented with penicillin and streptomycin (Invitrogen) and gentamycin (Invitrogen). Then, colons were cut in small pieces and incubated with gentle shaking for 30 min at 37°C in RPMI medium containing antibiotics, 5 mM EDTA (Roth), 3% FCS (Invitrogen), and 0.145 mg/ml DTT (Sigma-Aldrich). For IEC isolation, the supernatant was filtered with 100-µM strainers and centrifuged, and the pellet was resuspended in 30% (vol/vol) Percoll (GE Healthcare). The top layer was isolated, and 100,000 cells were plated on collagen type I (GE Healthcare)–precoated 96-well plates and cultured overnight. For isolation of lamina propria cells, the residual colon pieces were incubated in RPMI medium containing antibiotics, 0.1 mg/ml Liberase (Roche), and 0.05% DNase (Roche) for 30 min at 37°C with gentle shaking. Then, the supernatant was filtered with 70-µM strainers, and pelleted cells were washed two times in RPMI medium containing 10% FCS. Myeloid cells were sorted with a flow cytometer (FACS Aria; BD) using anti–Ly-6G (eBioscience), anti-CD45 (eBioscience), and anti-CD11b (eBioscience), seeded on 96-well plates, and cultured overnight. The remainder of the lamina propria cells (mostly lymphocytes) indicated as others were cultured as well along with the myeloid and IECs.
8
Anticoagulant Comparison for Blood Sampling
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Different anticoagulants were tested for blood sampling. Commercial 9 mL tripotassium ethylenediaminetetraacetic acid (K3EDTA), lithium heparin (Li–heparin), and sodium citrate (Na–citrate) pre-filled polystyrene tubes (VACUETTE®) were used (all from Greiner Bio-One, Kremsmünster, Austria). Additionally, different volumes of 0.5 M EDTA (Carl Roth, Karlsruhe, Germany) in dH2O (200 µL, 1 mL, and 8 mL) were tested in 50 mL tubes. Per biological replicate, 30 mL of blood was used for investigation of the best anticoagulant. To test anticoagulants, subsequent cell isolation was performed via combined slow-speed centrifugation and density gradient centrifugation. Isolated PBMCs were resuspended in 5 to 10 mL RPMI-1640 medium (Gibco™, ThermoFisher Scientific, Waltham, MA, USA).
9
Femoral Fracture Histological Analysis
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Femora were dissected 5, 9, 13, 16, and 21 days after fracture and prepared for histological staining. In brief, bones were fixed in 10% paraformaldehyde (Merck Chemicals GmbH, Darmstadt, Germany) (in PBS) for 24 h and decalcified for 6 weeks in 20% ethylene diaminetetraacetic acid (EDTA, pH 7.3; Roth, Karlsruhe, Germany). The intramedullary pin was removed and the bone was dehydrated through a graded series of ethanol solutions. Afterwards, femora were embedded into paraffin and 5 µm sections were cut along the shaft axis. Three sagittal paraffin sections with a minimal intersection distance of 30 µm were selected for all stainings, respectively, and were deparaffinized and rehydrated before the staining procedures. After staining, overview images were scanned for further semi-quantitative analysis (TissueFAXSi plus System, TissueGnostics, Vienna, Austria; INST89/341-1FUGG).
10
SDS-PAGE Protein Analysis Protocol
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For SDS-PAGE analysis, precast 4–20% Bis-Tris gradient gels (Genscript, Picastaway, USA) in a Tris-MOPS running buffer was used. The molecular weight marker was Pierce™ Unstained MW marker (Thermo Scientific). Maximum volume of 20 µL was loaded in the wells on the SDS-PAGE gel. Solubilized extracts were mixed 14:5:1 with a 4xSDS sample buffer (50 mM Tris (VWR, Leuven, Belgium) pH 6.8, 2% SDS, 10% glycerol (VWR), 0.02% bromophenol blue (Sigma-Aldrich), 12.4 mM ethylenediaminetetraAcetic acid (EDTA, Carl Roth, Karlsruhe, Germany)) and 1 M dithiothreitol (DTT, Thermo Scientific), corresponding to a final DTT concentration of 50 mM. The samples incubated at 95 °C for 5 min to denature proteins. Running time for the gel was 45 min at 140 V. To visualize proteins, the gel was stained with Coomassie Brilliant Blue G250 (29 mM Coomassie Brilliant Blue G-250 (Sigma-Aldrich), 45% Ethanol (VWR), 10% Acetic acid (Fisher Scientific, Loughborough, UK)). Destaining of the gel was performed overnight with destain solution (8% Ethanol, 5% Acetic acid). Imaging of the gel was done using a ChemDoc MP Imaging System (Bio-Rad, Hercules, USA).
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