At the appropriate time points, the media was replaced with an equivalent volume of 50 mM EDTA (BioRad, Hercules, CA) and incubated for 30 minutes at 37°C, 5% CO2. The digested alginate from each well was individually centrifuged for 2 minutes at 1,500 rpm, and the cells obtained were resuspended in 300 µL of 0.5 mg/mL MTT (ATCC) solution in DMEM. The MTT solution was also placed on the cell monolayer post alginate digest. These solutions were then incubated for 4 hours at 37°C before the addition of the detergent reagent (ATCC) for an overnight incubation. The final absorbance was read at 570 nm using a Tecan Infinite 200 PRO spectrophotometer (Durham, NC). The MTT absorbance reading for the digest was divided by the sum of the MTT readings for the digest and monolayer in order to find the percent migration for each well. Qualitative microscopic analyses were also performed on the 2.5D platform prior to alginate digestion. Gels were observed under the microscope to track the detachment of cells from the underlying monolayer on the TCPS and migrating into the alginate. These qualitative measurements were used as safeguards of the quantitative MTT-based measurements, as well as to prevent false positives or false negatives for migration.
Ethylene diamine tetraacetic acid (edta)
Manufactured by Bio-Rad
Sourced in United States, Germany
EDTA is a common laboratory reagent used as an anticoagulant. It chelates divalent metal ions, such as calcium and magnesium, which are necessary for the coagulation process. This property makes EDTA useful for preventing blood clotting in samples collected for analysis.
Automatically generated - may contain errors
Lab products found in correlation
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)
Ethylene diamine tetraacetic acid (edta), by Carl Roth (3 mentions)
Urea, by Merck Group (2 mentions)
Glycine, by Avantor (2 mentions)
Temed, by Bio-Rad (2 mentions)
Tris buffer, by Avantor (2 mentions)
Deoxycholic acid, by Avantor (2 mentions)
Detergent reagent, by American Type Culture Collection (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by American Type Culture Collection (1 mentions)
Hepes, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Eclipse e600, by Nikon (1 mentions)
Gel doctm xr image system, by Bio-Rad (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Gelatin, by Merck Group (1 mentions)
Nonidet p 40, by Thermo Fisher Scientific (1 mentions)
20 protocols using ethylene diamine tetraacetic acid (edta)
1
Quantitative Cell Migration Assay
2
Kidney Tissue Fractionation Protocol
3
Quantifying Alveolar Bone Infiltration
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Monocyte and lymphocyte infiltration was determined as the number of cells in the alveolar bone biopsies, which were histologically prepared from all groups. Mouse maxillas were harvested following sacrifice and were prepared and examined using immunohistochemical staining, as previously described (14 (link)). Briefly, the maxillas were decalcified in 10% ethylene diaminetetraacetic acid (EDTA; Bio-Rad Laboratories, Inc.) for 14 days, and were subsequently embedded in paraffin. Samples were cut into 5 µm sections and analyzed using immunohistochemical staining. Both sides of the maxillas were investigated and five slides were used for each sample at 10 intervals. Cell counts were conducted using images captured using a camera attached to a light microscope (Nikon Eclipse E600; Nikon Corporation, Tokyo, Japan) in a square of 45×70 µm.
4
Native Gel Electrophoresis of ODN Samples
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Native gel electrophoresis experiments were performed on 20 % polyacrylamide gels containing TBE (8.9 mm Tris, 8.9 mm borate, 0.2 mm EDTA, from BIORAD) and 30 mm KCl, at room temperature, 120 V for 2 h. The ODN samples, annealed at 1.6 mm single strand concentration in 1.0 m K+ buffer, were diluted at 0.6 mm loading concentration just before the PAGE runs. Glycerol was added (10 % final) to facilitate sample loading in the wells. The bands were finally visualized by ethidium bromide staining in a Bio‐Rad Laboratories Gel DocTM XR+ image system.
5
Cell Migration Quantification in Alginate
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After three days, the medium was replaced with an equivalent volume of 50 mM EDTA (purchased from BioRad, Hercules, CA, USA) and incubated for 30 min at 37 °C and 5% CO2. The digested alginate from each well was individually centrifuged for 2 min at 1500 rpm, and the cells obtained were quantified using the commercially available, formazan-based WST assay as per the manufacturer’s instructions. Briefly, the cells were resuspended in WST solution diluted in medium (1:5 v/v). The WST–medium mix was also added to the well plates post-alginate digestion to quantify the cells that did not migrate into alginate. These WST solutions were then incubated for 4 h at 37 °C, followed by centrifugation to remove the cells. Absorbance was then measured at 570 nm using Tecan Infinite 200 PRO (Durham, NC, USA). The WST absorbance reading of the alginate digest was divided by the sum of WST readings of the digest and well plate to calculate the percent migration for each well. Qualitative microscopic analyses were also performed on the sandwich cultures prior to alginate digestion to track and observe the cells that migrated into alginate.
6
Gelatin Zymography for Metalloprotease Detection
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Ten microgram of venom protein samples was run on polyacrylamide gels co-polymerized with 0.12% gelatin (Sigma-Aldrich) as a substrate [65 (link)]. The gel was washed in a 2.5% Triton X100 solution for 1 h and later for 30 min in a 0.05 M Tris-HCl (pH 7.4) buffer and finally incubated for 2–3 h in a 0.2 M NaCl, 0.005 M CaCl2, Triton X-100 0.002% (v/v), 0.001 M cysteine and 0.05 M Tris-HCl (pH 8) buffer. To confirm the presence of metalloproteases, venom samples were incubated for 30 min with EDTA (Bio-Rad) at 10, 50, and 100 mM concentrations in a buffer solution without CaCl2.
7
Western Blotting Protein Detection Protocol
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Western blotting was performed as previously described (41 (link), 42 (link)). In brief, cells were collected and lysed in RIPA buffer (50 mM Tris [MilliporeSigma, T6066] pH 7.5, 150 mM NaCl [Fisher Chemical, S271-500], 1 mM EDTA [Bio-Rad, 161-0729], Nonidet P-40 [Thermo Fisher Scientific, 85124], 0.1% SDS [MilliporeSigma, L3771-500G], and protease inhibitor cocktail [MilliporeSigma, P8340]), which disrupts membrane-associated proteins. Cell lysates were clarified by centrifugation for 10 minutes, 12,000g at 4°C, and boiled at 100°C for 10 minutes with SDS loading buffer containing 2-Mercaptoethanol. Treated samples were resolved on 10% SDS-PAGE gels, transferred to PVDF membranes (Bio-Rad, 162-0177), and probed with primary antibodies.
8
Purification and Characterization of E. coli Asparaginase
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Culture media ingredients, tryptone, and yeast extract were from Difco Laboratories, India. Tris buffer, glycine, IPTG, sodium dodecyl sulfate, PMSF, and deoxycholic acid were from Amresco, USA. Ammonium persulfate, acrylamide, bis-acrylamide, E. coli asparaginase, L-arginine and Urea were from Sigma Chemicals, USA. DEAE-sepaharose and S-200 gel filtration matrix was from GE Healthcare, Sweden. TEMED, EDTA, bromophenol blue from Biorad, USA. Coomassie brilliant blue R-250 and ampicillin from USB Corporation, Cleveland, Ohio. Glucose, NaCl, Nessler's reagent, and other chemicals were from Qualigen, India.
9
Biochemical Reagent Preparation Protocol
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Tris buffer, glycine, sodium dodecyl sulphate, phenylmethylsulfonyl fluoride (PMSF), and deoxy cholic acid were from Amresco (USA). Ammonium persulphate, acrylamide and bis-acrylamide, TEMED and EDTA, were from Biorad (USA). Coomassie Brilliant Blue R-250, Nonidet-P40, DNase I, Triton X-100, Bovine Serum Albumin (BSA), Congo red, IPTG, urea, proteinase-K, thioflavin-T, ampicillin and kanamycin were from Sigma-Aldrich (USA). SDS-PAGE molecular weight marker was purchased from Fermentas Thermo Scientific (USA). Glucose and NaCl from REASOL (Mexico), all other reagents were from J.T. Baker (USA). Paraformaldehyde, glutaraldehyde, osmium tetraoxide, Epon/Araldita, uranyl acetate and citrate were from Electron Microscopy Sciences (USA).
10
Quantifying SARS-CoV-2 Spike Binding
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HEK293T cells were transfected with 1000 ng S-C9 or WT S constructs with PEI. After 36 hours after transfection, cells were washed with PBS (MilliporeSigma, D5652-1L), detached with PBS containing 5 mM EDTA (Bio-Rad, 161-0729) for 10 minutes, washed twice with cold PBS plus 2% FBS, and incubated with soluble ACE2-hFc proteins (10 μg/mL, a gift from Jason McLellan’s lab at the University of Texas at Austin) for 2 hours. After 3 washes with PBS plus 2% FBS, cells were incubated with FITC-conjugated anti–human IgG (1:200, MilliporeSigma, F0257) secondary antibodies for 1 hour. Cells were washed 3 times with cold PBS plus 2% FBS and fixed with 3.7% formaldehyde for 10 minutes and analyzed by flow cytometry.
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