3 Materials and Methods
3.3 Methods
3.3.8 Degradations process of MgAg1% sticks
3.3.8.5 Determination of the silver concentrations in the degradation medium…
kit NANOCOLOR Silver 3 test using a photometric procedure determination. Ag+ could be measured between 0.20-3.00 mg/l Ag+, as Ag ions react with an indicator to form a blue dye. Briefly, for the determination of Ag+ concentrations in degradation medium the following was done:
A stock solution of 1 mmol/l AgNO3 was prepared and a calibration standard curve of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.8 mmol/l was prepared (Fig. 9).
Sterile MgAg 1% sticks were incubated in different volumes of medium (1, 3 and 10 ml) or NaCl without adding any other supplements, in addition to one tube of medium as negative control. They were incubated in a water bath at 37°C with shaking process for 25 days. The test was applied according to the manual manuscript with slight modifications. 28 µl NANOCOLOR Silver 3 were added per well of a 96-well plate. 10 µl of the NANOCOLOR Silver 3 R2 was added. Then 80 µl of tested sample was added and all the reagents were shaken. Afterwards, 10 µl NANOCOLOR Silver 3 R3 was added per well, then it was mixed again and incubated for 10 min. The samples were in duplicate number. The measurements were performed on day 5, 10, 15, and 25 using a photometer with a wavelength of 620 nm.
Materials and methods
y = 0.0704x + 0.0512 R2 = 0.9956
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
0 0.2 0.4 0.6 0.8 1 1.2
Concentration of Silver nitrate solution (m m ol/l)
Optical density
Fig. 9: Calibration standard curve for the determination of Ag+ concentrations, n=6.
- Calculated from the measured calibration function of the optical standards.
- Densities of total hardness measurement and the measurement of silver.
- Y = optical density, X = salt concentration, R2 = coefficient of determination (> 0.95)
3.3.8.6 Determination of the magnesium and calcium concentrations in the degradation media
The Mg++ and Ca++ concentration in the degradation media were measured by the test kit NANOCOLOR hardness 20 using a photometric procedure. The test is based on measuring the total hardness of the medium, which is primarily maintained by Mg++ and Ca++, but is also determined by barium and strontium ions. The color intensity depends on the total hardness of the surrounding medium. The calcium content can be obtained from the measured values of total hardness and the Mg++
content can be calculated.
A stock solution of MgCl2 was prepared (10 mmol/l) and a calibrations standard curve of 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4 and 5 mmol/l was prepared (Fig. 10). The samples were prepared from the degradation media by incubating MgAg1% sticks in different volumes of 10, 3 and 1 ml of medium or NaCl. One tube of pure medium or pure NaCl served as negative control. Subsequently, 6 µl of tested samples were added in each well of a 96-well plate, and then 180 µl of NANOFIX hardness 20 R2 was added per sample and 6 µl of hardness 20 R3 solution was added per sample. It was mixed
Materials and methods
y = 0.0542x + 0.1464 R2 = 0.991
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
0 1 2 3 4 5 6
Concentration of magnesium chloride (mmol/l)
Optical density
by pipetting up and down. The samples were in duplicate number. The samples were measured at 570 nm.
Samples of higher concentrations above the calibration curve were diluted. The measurements were performed at day 5, 10, 15 and 25.
Fig.10: Calibration standard curve for the determination of Mg++ concentrations, n=6.
- Calculated from the measured calibration function of the optical standards - Densities of total hardness measurement and the measurement of magnesium.
-Y = optical density, X = salt concentration, R2 = coefficient of determination (> 0.95).
Materials and methods
3.3.9 Biocompatibility tests
3.3.9.1 Measurement of cell viability and proliferation 3.3.9.1.1 MTS assay
Cell viability and proliferation for each cell type was evaluated using a modified MTS test (3 - (4, 5-dimethylthiazol -2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) - 2H-tetrazolium]) according to the manufacturer’s protocol. It is a colorimetric test for measuring the enzyme activity depending on the reduction equivalent of the tetrazolium salt. By using enzymatic activities of viable cells, viable cells reduce the yellow water soluble inner salt MTS to the brown water soluble at 490 nm absorbing formazan compound. Phenazine ethosulfate (PES) serves as an electron coupling reagent. To obtain a growth curve, 10.000 cells/well were plated in a 96-well plate up to confluence (5 days), during incubation with different concentration, of AgNO3 and degradation media of Mg/Ag1% sticks for 24 hours as mentioned before in 3.3.7. The test was applied according to the manufacturer’s protocol, the reagent solution was diluted in 1:4 in culture medium and 100 µl of this solution was added to each well.
The measurement of the extinction at a wavelength of 490 nm was performed after one hour of incubation at 37°C and 5 % CO2. Optical density of formazan was measured in this assay.
3.3.9.1.2 Neutral red assay
Cell viability and proliferation for each cell type were evaluated using the neutral red assay according to the manufacturer’s protocol. It is a colorimetric test for detecting cytotoxicity by inclusion of neutral red dye in lysosomes of vital cells. Living cells are coloured red, while dead cells are not stained as they do not have intact lysosomes any more. To obtain a growth curve, cells were plated and treated the same way as mentioned in 3.3.7; then the test was applied according to the manufacturer’s protocol:
The reagent solution was diluted 1:10 with the culture medium. 200 µl of the neutral red solution was added per well and the cells were incubated for 3 hours at 37°C and 5 % CO2. The cells were washed, then 100 µl neutral red solution 1 (1 % CaCl2, 0.5
% formaldehyde) were added for fixation for few minutes and were discarded.
Materials and methods
Afterwards, 100 µl neutral red solution 2 (1 % acetic acid, 50 % ethanol) were added to extract the incorporated neutral red dye. The plates were placed on an orbital shaker for 10 min. The measurement of the extinction with a wavelength of 570 nm was performed after one hour of incubation at 37°C and 5 % CO2. Neutral red optical density was measured in this assay.
Materials and methods
3.3.9.2 Measurement of metabolic activities
3.3.9.2.1 Measurement of the succinate dehydrogenase (SDH) activity in the supernatant
SDH binds to the inner mitochondria membrane of mammalian and many bacterial cells. It takes an active part in the citric acid cycle and electron transport chain.
Succinate is the most efficient energy source, so the SDH activity assay is an important method for measuring of metabolic activities (SAMOKHVALOV et al., 2004). In many cell types, mitochondria are the primary source of reactive oxygen species (ROS) (CADENS and DAVIES, 2000). The two major superoxide producing enzymes are considered to be the respiratory chain complexes nicotinamide adenine dinucleotide (NADH) and cytochrome C oxidoreductase (SUN and TRUMPOWER, 2003). Succinate oxidoreductase was discovered in 1909 in aerobic cells (THUNBERG, 1909). The enzyme has several interesting properties, for example that SDH is a membrane bound dehydrogenase linked to the respiratory chain and a member of the Krebs cycle (Fig. 11), beside its activity which is modulated by several activators and inhibitors. SDH is a complex enzyme containing nonheme iron, acidlabile sulfur, and covalently bound flavin adenine dinucleotide (FAD) (HEDERSTEDT and RUTBERG, 1981).
Fig. 11: Citric acid cycle (HEDERSTEDT and RUTBERG, 1981).
SDH
Materials and methods
According to MRACEK et al. (2009), SDH was determined with some modifications spectrophotometrically in isolated mitochondria. The cells were plated and treated in the same way as mentioned in 3.3.7, and then the protocol of measuring SDH was done as follow:
Cells were washed twice with PBS, 100 µl of Aqua bidest. was added in each well.
The cells were lysed for 10 min by heat and cold, then were sonicated for 15 seconds and were centrifuged at 2000 x g at 4 °C for 15 min. Afterwards, the supernatants were used for protein determination and 50 µl SDH reaction solution was added per well. The measurements of the extinction were performed at a wavelength of 340 nm at different time points (zero time, 10 min, 20 min and 30 min). Enzyme activities were expressed as nmol X min-1/mg protein-1, using the molar absorption coefficient (e550 = 19.6 mM-1 cm).
3.3.9.2 Measurement of the pyruvate kinase (PK) activity in the supernatant Pyruvate is the end product of the glycolysis. Under aerobic conditions pyruvate enters the mitochondria and catalyzes the conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) producing NADH and CO2. Acetyl-CoA subsequently enters into the Krebs (citric acid) cycle, providing energy adenosine triphosphate (ATP) to the cell (Fig. 12). This reaction is catalyzed by the enzyme-coenzyme complex pyruvate dehydrogenase (PDH) (HARRIS et al., 2002). PDH activity is under the control of pyruvate dehydrogenase kinases (PDKs). Under hypoxic conditions, conversion of pyruvate to lactate occurs. Lack of PK leads to a decrease in the glycolysis process.
According to IORI et al. (2008), cells were plated and treated in the same way as described in 3.3.7, and then the protocol of measuring pyruvate was done as follow:
The medium was removed from the cells, they were washed with PBS. 100 µl PBS were added and cells were lysed for 10 min by heat and cold, and then sonicated for 2 min and cells were centrifuged at 2000 × g for 15 min at 4°C. The supernatants were used for protein determination (BRADFORD, 1976), The cells were collected using 250 μl of 100 mM triethanolamine, 0.5 mM EDTA/Na+ buffer (pH 7.6), supplemented with protease inhibitors. The enzyme assay starts by adding 100 μl of PK reaction solution. PK activity was estimated by a modification of the spectrometric method (GUTMANN and BERNT, 1974). The PK activity was measured as the
Materials and methods
change in absorption of NADH at 340 nm (25 °C) due to the coupled conversion of pyruvate to lactate catalyzed by lactate dehydrogenase (LDH). The measurements were performed on different time points (zero time, 10 min, 20 min and 30 min).
Fig. 12: Pyruvate kinase pathway (YAISH, 2009)
3.3.9.2.3 Determination of the protein content
The protein content of the cells was measured using Bio-Rad assay based on the method of Bradford. Bio-Rad is a dye binding assay, in which a differential colour change of a dye occurs in response to various concentrations of protein. The maximum absorbance of an acidic solution of Coomassive ® Brilliant Blue G-250 dye shifts when binding to protein. Coomassive blue dye binds to primary basic acids and aromatic amino acids residues especially arginine. Subsequent measurements with a spectrophotometer and comparing the results to a standard curve provide a relative measurement of protein concentration.
The test was done as follow:
Frozen samples were thawed and 160 µl of standard calibrations were added in each well of a 96-well plate, then 159 µl PBS was added to each sample (1 µl). Afterwards, 40 µl Bio-Rad reagents were added for the whole plate and all wells were mixed well 10 times. The plate was left at room temperature for 5-60 min. The measurements of the extinction took place at a wavelength of 570 nm.
Materials and methods
3.3.9.3 Histochemistry analysis
To evaluate SDH activity by histochemistry (SELIGMAN and RUTENBURG, 1951;
BROUILLET et al., 1998) frozen teat sections were cryosectioned at 8 µm at a temperature of -20°C. The udder tissue sections were transferred to glass slides and air-dried for 60 min before histochemical staining. Each tissue section was then incubated for 1 h at 37°C in a dark and humidified chamber in SDH phosphate buffer containing succinic acid (as a substrate) and nitroblue tetrazolium (SELIGMAN and RUTENBURG, 1951; TANJI and BONILLA, 2001; KIYOMOTO et al., 2008). After this procedure, the slices were rinsed with Aqua bidest. to stop the chemical reaction.
The slides were then cover slipped. Endogenous SDH activity resulted in dark blue diformazan deposits from the NBT reduction through succinate oxidation. No blue deposits are formed in the absence of succinate substrate or in the presence of 3NP in the incubation. To quantify SDH, an image of each section was captured using camera connected to an optical microscope. The protocol was applied on cell culture samples by culturing and treating in the same way as mentioned before in 3.3.7.
The medium was removed and cells were washed twice with PBS. 100 µl of histochemistry buffer was added and incubated for 4 h at 37°C and 5 % CO2. The measurements were performed using a spectrophotometer with a wavelength of 570 nm.
Materials and methods
3.3.10 Measurement of biomarkers of inflammatory reactions 3.3.10.1 Preparation of the udder tissue for PGE2 measurements
The teats were cut in small pieces of approximately 1 cm3 (25-30 mg) using a biopsy punch and were transferred in polypropylene tubes over crushed ice. A mixture of 5 mg indometacin + 5 ml DMSO (dimethylsufoxide) + 1 ml PBS was added and the samples were mixed by an ULTRA–TURRAX for 30 min, followed by centrifugation at 3000 x g for 10 min. The supernatants were collected for the PGE2 measurement.
3.3.10.2 Measurement of PGE2 in the udder tissue supernatant
The PGE2 concentration in the culture medium supernatant was measured by a competitive enzyme immunoassay. This assay is based on the forward sequential competitive binding technique, in which PGE2 competes with horseradish peroxidase (HRP) - labelled PGE2 for a limited number of binding sites on a mouse monoclonal antibody. PGE2 in the sample is allowed to bind to the antibody in the first incubation.
During the second incubation, HRP- labelled PGE2 binds to the remaining antibody sites. The amount of PGE2 tracer that is able to bind to the monoclonal antibody is inversely proportional to the concentration of PGE2 in the sample. This antibody-PGE2 complex binds to goat polyclonal anti-mouse IgG that has been previously attached to the well of the assay kit. The plate is washed to remove any unbound material, and a substrate solution is added to the wells to determine the bound enzyme activity. The product of this enzymatic reaction has a distinct yellow colour and absorbs strongly at 412 nm, when the colour development has been stopped.
The intensity of this colour is determined spectrophotometrically and is proportional to the amount of PGE2 tracer bound to the well, which is inversely proportional to the amount of free PGE2 present in the sample during the incubation. A standard curve was performed in each ELISA assay. The percentage of binding was used to establish a calibration curve. The measurements for the extinction of each sample were put in relation to these curves and the PGE2 concentration was calculated. The samples were usually measured in duplicates and the following two controls were included in each. One of the samples was stimulated by the addition of 5 mL lipopolysaccharide (LPS) working solution (500 ng/ml LPS, final concentration) while
Materials and methods
the other control was a pure medium. The supernatant was collected 24 h later and stored at -20°C until determination.
3.3.10.3 Measurement of bovine and mouse TNF-alpha in the culture medium supernatant
The TNF-alpha concentration in the culture medium supernatant was measured.
Briefly, the wells of the assay plates were preincubated with a monoclonal goat anti-mouse or bovine TNF-alpha antibody which binds any TNF-alpha of the sample (bovine or mouse). The bound TNF-alpha is detected using a biotinylated goat anti-mouse or bovine TNF-alpha antibody, and addition of TMB-substrate (tetramethylbenzidine) leads to a color reaction which stopped by using stop solution H2SO4 (2N). TNF-alpha was measured spectrophotometrically at 450 nm. The intensity of the colour is proportional to the concentration of TNF-alpha in the sample.
3.3.10.4 Measurement of IL- 6 in the culture medium supernatant
The IL-6 concentration in the culture medium supernatant was measured similarly to the TNF-alpha concentration with an enzyme immunoassay. Briefly, the wells of the assay plates were preincubated with rat anti-mouse IL-6 antibody, which binds any IL-6 of the sample. The bound IL-6 is detected utilizing a biotinylated goat anti-mouse IL-6 (detection antibody) and TMB-substrate. The colour reaction was stopped using stop solution H2SO4 (2N) andwas measured spectrophotometrically at 450 nm. The intensity of the colour is proportional to the concentration of IL- 6 in the sample.
3.3.10.5 Measurement of IL-1 beta in the culture medium
The IL-1 beta concentration in the culture medium supernatant was measured similarly to the IL-6 concentration with an enzyme immunoassay using the same capture and detection antibody.
All those assays are established methods at the Institute of Pharmacology, Toxicology and Pharmacy, Hannover, Germany.
Materials and methods
3.3.11 Detection of antibacterial activity
3.3.11.1 Bouillion dilution test and cultivation of bacteria in petri dishes
Bouillion dilution test and cultivation of bacteria in petri dishes were done by the Milchtierherden-Betreuungs- und Forschungsgesellschaft mbH (MBFG). The aim of the tests is to determine the effect of Mg-Ag-NaCl solutions on the growth of bacteria (E. coli and S. aureus).
Briefly, MgAg1% sticks were incubated in 5 ml NaCl solution in a water bath for 15 days and the degrading supernatants (MgAg1% sticks incubated in NaCl) were used for the bouillion dilution test and the cultivation of bacteria (E. coli and S. aureus).
Firstly, bouillion dilution test was done by preparing a bacterial suspension by incubating about 2 E. coli and S. aureus colonies (GK) in 100 ml buffered peptone water at 37°C for 24 hours on the shaker. The bacterial dilution series was770 X 105, 910 X 105, 100 X106 and 210 X106 KbE/ml. Then 2 test tubes were filled by 5 ml of bacterial suspension, then degrading supernatants (MgAg1% sticks incubated in NaCl) were added to one of the tubes, and the other one serves as a negative control. Later, the number of bacterial colonies was determined.
Secondly, E. coli and S. aureus colonies were cultivated in petri dishes. Afterwards, the bacterial colonies were incubated with the degrading supernatants (MgAg1%
sticks incubated in NaCl), and the number of bacterial colonies was determined.
3.3.11.2 Brilliant black reduction (BRT- MRL Screening test)
The BRT was first described by KRAACK and TOLLE (1967). The test medium is a mixture of nutrients, test bacteria Geobac. Stearothermophilus var. calidolactis C953 (B. stearothermophilus), brilliant black and other supplements which help to improve detection sensitivity towards chosen inhibitors (Fig. 13). Penicillin G serves as a positive control and milk as a negative control.
Materials and methods
Fig.13: The BRT- MRL screening test cavities
MgAg1% sticks were incubated with different volumes of DMEM medium (10, 3 and 1 ml). One tube of medium serves as a negative control, they were incubated in a water bath for 5 days. In addition, different concentrations of AgNO3 solution (control, 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mmol/l) were incubated with DMEM medium in test tubes without adding any supplements in a water bath for 5 days. 100 µl of each sample were added per well. Adhesive tapes were chosen to cover the wells during incubation for 5-6 hours at 65°C in a special incubator (Fig.
14).
Fig.14: Incubator of BRT- MRL Screening test
During the incubation time, the growing test bacteria shift the redox indicator (brilliant black) to its yellow or colourless reduction stage through the division of double azocompounds. Thus test medium changes from blue to yellow or colourless, if inhibitors are not present in the sample, therefore, the sample has no antibacterial effect. While the growth of bacteria will be minimal or non-existent, when there will be no reduction of the colouring agent or to a very small degree and the test medium will
Cavities containing test medium + other supplements
Materials and methods
remain blue, if the inhibitors are present in the medium, therefore, the sample has antibacterial effect.
3.3.12 Statistical analysis
Values are means ± SD. A randomized block design was used in all of the experiments. Samples were usually measured at least in duplicates. The experiments were performed at least 4-6 times.To enable better comparison of the experiments, results are expressed in optical density and percent in comparison to the negative control.
For statistical calculation of differences in the viability of isolated perfused udder (glucose consumption, lactate production and LDH activity), Two-way ANOVA test was performed. For the biocompatibility tests (MTS, neutral red, SDH, PK, IL-1 beta, IL-6 and TNF-alpha), One-way ANOVA test was performed followed by a Dunn’s Multiple comparison test. For the degradation process of MgAg1% sticks in dry off period secretion and in the teats of isolated bovine udder, t-test was performed. For silver and magnesium concentrations in degradation medium, Two-way ANOVA test was performed.
Statistical calculations were performed with GraphPad Prism® 5.03 (GraphPad Software Inc., La Jolla). P values < 0.05 were considered statistically significant.
Results
4. Results
4.1 Establishment of mammary cell culture 4.1.1 Isolation of primary mammary cells
Mammary cells were enzymatically and mechanically isolated and taken into culture.
Both primary mammary fibroblasts and primary mammary epithelial cells were morphologically distinct (Fig. 15 A, B). Each cell type exhibited the typical morphological characters. The fibroblasts showed a spindle shaped morphology and the epithelial cells grew in a cobblestone-like pattern. The purity of each cell
Both primary mammary fibroblasts and primary mammary epithelial cells were morphologically distinct (Fig. 15 A, B). Each cell type exhibited the typical morphological characters. The fibroblasts showed a spindle shaped morphology and the epithelial cells grew in a cobblestone-like pattern. The purity of each cell