The total mucin content was determined following the method described in54 (link) by equilibrating the collected chip mucus samples with Alcian Blue stain (ThermoFisher, Cat. no. 88043) for 2 h, followed by centrifuging the resulting precipitant at 1870 g for 30 min, then 2 X wash/spin cycles with a washing solution composed of 40% ethanol, 0.1 mol/L acetic acid, and 25 mmol/L magnesium chloride. The stained mucin pellets were then dissociated in 10% sodium dodecyl sulfate solution (Sigma, Cat. no. 71736), and the absorbance was measured with a microplate reader (Syn-Q55 Synergy HT, BioTek) at 620 nm. Mucin concentrations were obtained based on a curve fitted to mucin standards developed from submaxillary gland mucin (Sigma, Cat. no. M3895) serially diluted from 0 – 500 μg/ml, and Alcian Blue stained as described above.
Sodium dodecyl sulfate solution
Manufactured by Merck Group
Sourced in United States
Sodium dodecyl sulfate (SDS) is a widely used anionic surfactant solution. It is commonly used in biochemical and molecular biology applications, such as protein electrophoresis and Western blotting, to solubilize and denature proteins.
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Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
Lysozyme from chicken egg white, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Ultrapure distilled water, by Thermo Fisher Scientific (1 mentions)
Tris hci, by Thermo Fisher Scientific (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Fibronectin, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem culture medium, by Thermo Fisher Scientific (1 mentions)
Versamax, by Molecular Devices (1 mentions)
Leucine standard, by Merck Group (1 mentions)
Tnbs solution, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
8 protocols using sodium dodecyl sulfate solution
1
Quantifying Mucin Content Using Alcian Blue Assay
2
Magnetic Bead-based Oligonucleotide Capture
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Triton X-100 (Sigma-Aldrich), Tris-HCI (1
M, pH 8.0, Invitrogen), EDTA (0.5 M, pH 8.0, Invitrogen), lysozyme
from chicken egg white (Sigma-Aldrich), streptavidin conjugated magnetic
beads (1 μm, OceanNanoTech), sodium chloride (Sigma-Aldrich),
20× SSC (IBI Scientific), sodium dodecyl sulfate solution (10%,
Sigma-Aldrich), UltraPure distilled water (Invitrogen), bovine serum
albumin (>98%, Sigma-Aldrich), 1× phosphate buffered saline
(Sigma-Aldrich),
Biotinylated single-stranded oligonucleotides with C12 linker between
biotin and oligonucleotides (Eton Bioscience).
EC1:5′-biotin-C12-CTGCGGGTAACGTCAATGAGCAAA-3′
EC2:5′-GGTATTAACTTTACTCCCTTCCTC-C12-biotin-3′.
M, pH 8.0, Invitrogen), EDTA (0.5 M, pH 8.0, Invitrogen), lysozyme
from chicken egg white (Sigma-Aldrich), streptavidin conjugated magnetic
beads (1 μm, OceanNanoTech), sodium chloride (Sigma-Aldrich),
20× SSC (IBI Scientific), sodium dodecyl sulfate solution (10%,
Sigma-Aldrich), UltraPure distilled water (Invitrogen), bovine serum
albumin (>98%, Sigma-Aldrich), 1× phosphate buffered saline
(Sigma-Aldrich),
Biotinylated single-stranded oligonucleotides with C12 linker between
biotin and oligonucleotides (Eton Bioscience).
EC1:5′-biotin-C12-CTGCGGGTAACGTCAATGAGCAAA-3′
EC2:5′-GGTATTAACTTTACTCCCTTCCTC-C12-biotin-3′.
3
Protein Adsorption on Scaffold Materials
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Protein adsorption test was carried out in accordance with the procedure described previously.10 (link) In brief, wetted disc-shaped scaffolds, approximately 2 mm thick and 5 mm in diameter, were placed in a 96-multi-well plate and incubated for 3 hours at 37°C on a shaker (125 rpm, Sky Line DTS-4, ELMI Ltd., Riga, Latvia) in human blood plasma (collected from a healthy volunteer after obtaining written informed consent) or in human protein solutions (1 mg/mL) prepared in PBS (Pan-Biotech GmbH, Aidenbach, Bavaria, Germany): albumin, fibrinogen (Sigma-Aldrich Co.), fibronectin (BD Biosciences, San Jose, CA, USA). Empty wells of polystyrene (PS) 96-multi-well plate with protein solutions were treated as a test control to check unspecific binding of proteins to PS. After 3 hours' incubation, protein solutions were discarded, scaffolds were moved into new wells of 96-multi-well plate, washed four times with PBS, and incubated in 1% sodium dodecyl sulfate solution (Sigma-Aldrich Co.) for 1 hour on the shaker (230 rpm) to recover proteins adsorbed to the samples. Protein concentrations in the collected solutions were assessed using the Lowry method as described previously.10 (link) Results were expressed as the quantity of adsorbed proteins (μg) per 1 cm2 of the scaffold.
4
Decellularized Tracheal Graft Preparation
5
Evaluating Mitochondrial Activity in HaCaT Cells
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After the supernatant for cytokine analysis was collected, HaCaT cells, plated in 96-well microplates, were incubated with methyl tetrazole formazan (MTT, 5 mg/ml; (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 20 % (v/v) in DMEM culture medium (Eagle's medium modified by Dulbecco, Gibco) for 3 h at 37 °C in a humidified atmosphere containing 5 % CO2 in order to evaluate mitochondrial activity. The formazan precipitates were dissolved by adding 100 μl of 10 % (w/v) sodium dodecyl sulfate solution (Sigma-Aldrich, St. Louis, MO, USA) overnight, and, after homogenization, optical density was read at 570 nm on a microplate reader (Versamax, Molecular Devices, Sunnyvale, CA, USA). The experiments were performed with three cell donors in duplicate, and cells grown under ideal conditions (without stimulation) were considered to be the controls. Data were recorded as relative units and compared with the control group.
6
Determination of Degree of Hydrolysis in Whey Protein Hydrolysate
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The DH was measured using the 2,4,6 trinitrobenzene sulfonic acid (TNBS) method [8 (link)]. A 50 mL sample of a 0.02% protein solution was prepared by dissolving 1 g of WPH powder in distilled water. Protein solutions were stirred for 30 min to ensure that the powder was fully dissolved. Then, 1 g of the protein solution was added to 9 g of 1% (w/w) sodium dodecyl sulfate (SDS) solution (Sigma Aldrich, St. Louis, MO, USA). In test tubes, a 0.1 mL aliquot of protein solution, 2 mL of phosphate buffer (pH 8.2), and 2 mL of 0.1% (w/w) TNBS solution (Sigma Aldrich, St. Louis, MO, USA) were added. A leucine standard linear curve was obtained by diluting 530 mg/L leucine standard (Sigma Aldrich, St. Louis, MO, USA) in 1% SDS (w/w) to obtain 42.4, 84.8, 127.2, 169.6, and 212 mg/L amino nitrogen leucine standards. All tubes were vortexed and placed in the water bath at 50 °C for 60 min. The reaction was stopped by adding 4 mL of 0.1 N HCL (Chemicals, Gibbstown, NJ, USA) to each tube. Standards and samples were measured at 340 nm using a UV/VIS spectrophotometer (Metash Instruments Inc., Shanghai, China) against a 1% SDS blank solution. The leucine standard curve was plotted, then calculations were carried out accordingly.
7
Cell Viability Assay with BCT-100
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Briefly, cells (5000/well) were incubated with different activity units of BCT-100 with/without cisplatin or pemetrexed. Cells incubated with medium only served as a negative control. Following incubation for 72 h, cells were fixed for 10 min with 4% formaldehyde (Sigma-Aldrich, St. Louis, Missouri, United States) in phosphate-buffered saline (PBS) and stained for 10 min with 0.05% crystal violet. The plates were washed for 4 times with tap water and then the cells were lysed with 1% sodium dodecyl sulfate (SDS) solution (Sigma-Aldrich). Absorbance (595 nm) was measured using a microplate reader Fluo Star Optima (Bmg Labtec GmbH, Ortenberg, Germany) [12 (link)].
8
In Vitro Cytotoxicity Assessment of Sgc8-c-carb-da, Sgc8-c, and Dasatinib
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Cells (5 × 104 cells/well) were seeded in a 96-well plate and cultured for 48 h at 37 °C in 5% CO2, with different concentrations of Sgc8-c-carb-da , Sgc8-c, or dasatinib, ranging from 400 to 80,000 nM. In addition, cells were incubated with 20% DMSO (Sigma-Aldrich) and culture medium as controls. Then, the medium was removed, and cells were washed with 1X phosphate buffered saline (PBS), and 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) was added to each well. The plates were incubated for 4 h at 37 °C in 5% CO2. To dissolve MTT crystals, a 20% sodium dodecyl sulfate (SDS) solution (Sigma-Aldrich) was added to the plates and further incubated overnight at room temperature in dark conditions. Finally, the plates were read at 570 nm to determine the optical density (OD) in each well. Cell cytotoxicity (%) was calculated using the following formula: [(OD in the studied condition—OD with DMSO)/(OD in control medium—OD with DMSO)] × 100. The half maximal inhibitory concentrations (IC50), defined as the concentrations that induce 50% cytotoxicity, were determined from the viability vs. concentration curves using the software GraphPad8 (version 8.0.1).
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