The exopolysaccharides (EPS) were precipitated from the supernatant of FM at 48 h of fermentation. Samples were centrifuged (3600× g, 60 min, 10 °C), and the supernatants were recovered. Afterwards, trichloroacetic acid solution (20% v/v, Sigma-Aldrich) was added to the supernatants, which were incubated for 2 h at 4 °C. Precipitated proteins were removed by centrifugation (3600× g, 60 min, and 10 °C). Next, the supernatants were treated with two volumes of cold ethanol, followed by 12 h of incubation at 4 °C. The EPS were recovered by centrifugation and posteriorly suspended in 1 mL of milli-Q water. The total EPS content was estimated for each sample by the phenol-sulfuric method using glucose as the standard [16 (link)].
Trichloroacetic acid solution
Manufactured by Merck Group
Sourced in United States
Trichloroacetic acid solution is a laboratory chemical used as a reagent in various analytical and research applications. It is a clear, colorless liquid with a pungent odor. The solution is typically available in different concentrations, depending on the specific requirements of the application.
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Lab products found in correlation
Gemini em microplate spectrofluorometer, by Molecular Devices (3 mentions)
Evans blue solution, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Evans blue dye, by Merck Group (1 mentions)
Tristar2 multimodal reader lb942, by Berthold Technologies (1 mentions)
Sulforhodamine b, by Merck Group (1 mentions)
Tris hcl, by Thermo Fisher Scientific (1 mentions)
L aspartate, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Malondialdehyde tetrabutylammonium salt, by Merck Group (1 mentions)
Guanidine hydrochloride, by Merck Group (1 mentions)
Butylated hydroxytoluene, by Merck Group (1 mentions)
Mgso4, by Carl Roth (1 mentions)
Cacl2, by Carl Roth (1 mentions)
2 thiobarbituric acid, by Merck Group (1 mentions)
2 4 dinitrophenylhydrazine dnph, by Merck Group (1 mentions)
Kh2po4, by Carl Roth (1 mentions)
Nahco3, by Carl Roth (1 mentions)
19 protocols using trichloroacetic acid solution
1
Exopolysaccharides Extraction and Quantification
2
Evaluating Blood-Brain Barrier Disruption
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BBB disruption was evaluated using Evans blue dye (Sigma–Aldrich) extravasation on the 72 h after tMCAO as described previously24 (link),26 (link). Briefly, 2% solution of Evans blue (Sigma–Aldrich) dissolved in sterile saline was injected through the rat tail vein at a dose of four mL/kg of body weight at 1 h before the rat were sacrificed. Before the rat brain samples were removed, rats were transcardially perfused with 100 mL of 0.9% cold saline to remove the intravascular dye. The brain sample were weighted, homogenized in 50% trichloroacetic acid solution (Sigma–Aldrich) diluted with saline and centrifuged at 12,000×g for 30 min. Then, the resultant supernatant was diluted 4-fold with ethanol and spectrophotometrically quantified at 620 nm to determine the extravasation of Evans blue dye (Sigma–Aldrich) in a ThermoFisher plate-reader (Waltham, MA, USA; excitation at 620 nm and emission at 680 nm).
3
Quantifying Blood-Spinal Cord Barrier Disruption
4
Sulforhodamine B Cell Viability Assay
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For this assay, 5 × 103 cells per well were plated in 96-well plates. Five days after seeding, the cell media was gently aspirated without touching adherent cellular layer and fixed with 50% trichloroacetic acid solution (Sigma-Aldrich, St. Quentin Fallavier, France) for 1 h at 4 °C. Following three washing steps with ddH2O, cells were stained with a solution of 0.4% sulforhodamine B (Sigma-Aldrich, St. Quentin Fallavier, France) in 1% acetic acid. Excess dye was removed by three washes with 1% acetic acid, and protein-bound dye was then dissolved in 10 mM Tris solution (pH 10.5). Absorbance was measured at 510 nm with a TriStar2 Multimodal Reader LB942 (Berthold Technologies). Data were plotted by using the SuperPlotsOfData webapp; the plots represent three independent experiments.
5
Aspartate Transcarbamylase Enzyme Assay
6
Quantifying Blood-Spinal Cord Barrier Disruption
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Evans Blue (EB) dye, 961 Da, was used as a tracer for assessing BSCB disruption. The EB extravasation assay was performed as previously described51 (link)–53 (link). Briefly, after perfusion, mouse spinal cords were weighed and placed in 50% trichloroacetic acid solution (Sigma). Following homogenization and centrifugation, the supernatant was diluted with ethanol (1:3) and loaded into a 96 well-plate in triplicate. The dye was measured with a spectrofluorometer (Gemini EM Microplate Spectrofluorometer, Molecular Devices) at excitation of 620 nm and emission of 680 nm54 (link). Calculations were based on external standards in the same solvent. The EB content in tissue was quantified from a linear standard curve derived from known amounts of the dye and was normalized to tissue weight (μg/g). All measurements were performed by two experimenters blinded to the experiment.
7
Gram-positive Enhancer Matrix Extraction
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L. lactis MG1363 cultured in M17 broth medium was harvested and resuspended in a 10% trichloroacetic acid solution (Sigma, St. Louis, MO, USA). After boiling for 30 min and washing five times with 0.01 M PBS, the resulting Gram-positive enhancer matrix (GEM) particles were diluted to 1 unit (U), where 1 U was defined as 2.5 × 109 GEM particles.
8
Evaluating Cell Viability after Drug Treatment
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Cell viability following drug treatment was accessed using Sulforhodamine B (SRB) assay and performed as previously described32 (link) with certain modifications. 10 000 cells/well were seeded in a 96-well plate and were allowed to incubate overnight in the humidified incubator. The following day, media was aspirated from each well and cells were incubated with fresh media containing different concentrations of the specified compound and further incubated at 37 °C incubator for 72 h.
After drug treatment, the medium was aspirated and cells were fixed with 10% trichloroacetic acid solution (Sigma-Aldrich, T6399) at 4 °C overnight. Plates were further processed as previously described.27 Dose–response curves were generated using GraphPad Prism 6 (La Jolla, CA, USA). Before obtaining GI50 values, curves were constrained at 100% on top and greater than 0% on bottom.
After drug treatment, the medium was aspirated and cells were fixed with 10% trichloroacetic acid solution (Sigma-Aldrich, T6399) at 4 °C overnight. Plates were further processed as previously described.27 Dose–response curves were generated using GraphPad Prism 6 (La Jolla, CA, USA). Before obtaining GI50 values, curves were constrained at 100% on top and greater than 0% on bottom.
9
Quantifying Blood-Brain Barrier Permeability
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Four mice per WT and KO at each time point were used for comparison. Under anesthesia with 4% isoflurane, Evans blue solution (Sigma, 2% in saline; 4 mL/kg) was injected via the tail vein. After circulation for 2 h, brains were extracted and dissected into hemispheres. Each hemisphere was homogenized (Tissue Tearor, 50 s; speed 35) in 1 mL (total volume) of 0.1 M PBS. The supernatant was collected and mixed with trichloroacetic acid solution (Sigma, 50% in saline). After centrifugal precipitation, the supernatant was extracted and the absorbance was measured at 610 nm with a spectrophotometer.
10
HPLC-grade Deionized Water Protocol
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HPLC-grade deionized water was applied during all experiments including Langendorff studies. All chemicals and reagents were of analytical grade and used without further purification.
NaCl, KCl, KH2PO4, MgSO4, CaCl2, NaHCO3, and dextrose were purchased from Roth (Karlsruhe, Germany). α-KG, 5-HMF, NALM, NASeLM, and glucose were supplied by CYL-Pharma (Lassnitzhöhe, Austria). Human albumin solution 20% was provided from Behring (Marburg, Germany). Acetonitrile, NH4CH3COO, 1-butanol, ethanol, HPLC-grade water, and HCl were obtained from Merck (Darmstadt, Germany). 2-Thiobarbituric acid, butylated hydroxytoluene, ethyl acetate, trichloroacetic acid solution, 2,4-dinitrophenylhydrazine (DNPH), malondialdehyde tetrabutylammonium salt, guanidine hydrochloride, and tris(hydroxymethyl) aminomethane were supplied by Sigma-Aldrich (Vienna, Austria).
NaCl, KCl, KH2PO4, MgSO4, CaCl2, NaHCO3, and dextrose were purchased from Roth (Karlsruhe, Germany). α-KG, 5-HMF, NALM, NASeLM, and glucose were supplied by CYL-Pharma (Lassnitzhöhe, Austria). Human albumin solution 20% was provided from Behring (Marburg, Germany). Acetonitrile, NH4CH3COO, 1-butanol, ethanol, HPLC-grade water, and HCl were obtained from Merck (Darmstadt, Germany). 2-Thiobarbituric acid, butylated hydroxytoluene, ethyl acetate, trichloroacetic acid solution, 2,4-dinitrophenylhydrazine (DNPH), malondialdehyde tetrabutylammonium salt, guanidine hydrochloride, and tris(hydroxymethyl) aminomethane were supplied by Sigma-Aldrich (Vienna, Austria).
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