3 Materials and Methods
3.2 Materials
3.2.6 Cell culture equipments .…
Heraeus-Kulzer, Hanau, Germany Thermo Electron LED GmbH, Langenselbold-Germany Zeiss, Oberkochen, Germany
AESCULAP-Werke TUTTLINGEN, Germany
Canon Deutschland GmbH, Krefeld, Germany
BÜCHI Laboratoriums-Technik AG, Essen, Germany
Eppendorf, Hamburg, Germany IKA WERKE, Breisgau , Germany
3.2.7 Magnesium-silver1% (MgAg1%) stick and silver nitrate
The MgAg1% alloy as shown in (Fig. 5) is made from 99% magnesium and 1% silver and has a weigh of 10 - 70 mg. It was produced with a length of 1 cm and a diameter of 0.2 cm (Institute of Materials Science, Leibniz University, Hannover, Germany).
Silver nitrate was purchased from Merck, Darmstadt, Germany.
Fig. 5: MgAg1 % stick
1 cm
Materials and methods
3.2.8 Materials and reagents
3.2.8.1 Materials and reagents for Bio-Rad assay
- BSA SIGMA-ALDRICH Chemie GmbH, Steinheim, Germany
- Bio-Rad Reagent BioRad Laboratories GmbH, Munich, Germany
3.2.8.2 Materials and reagents for measuring cytokines (Interlekin-1beta (IL- 1beta), interleukin- 6 (IL- 6), tumour necrotic factor (TNF-alpha)
- Mouse TNF-alpha/TNFSF1A DuoSet® R&D Systems, Inc., Minneapolis, USA ELISA Development System
- Mouse IL-1beta/IL-1F2 DuoSet ® R&D Systems, Inc., Minneapolis, USA ELISA Development System
- Mouse IL-6 DuoSet ® R&D Systems, Inc., Minneapolis, USA ELISA Development System
3.2.8.2.1 Equipments used for measuring cytokines
- 96- well micro plate R&D Systems, Inc., Minneapolis, USA - ELISA plate sealers R&D Systems, Inc., Minneapolis, USA
3.2.9 Materials and reagents used for measuring silver concentrations
NANOCOLOR® MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany Silver 3 test kits
3.2.10 Materials and reagents used for measuring calcium and magnesium Concentrations
- NANOCOLOR® MACHEREY-NAGEL GmbH & Co. KG, Düren, Hardness 20 test kits Germany
- Magnesium chloride Merck, Darmstadt, Germany
Materials and methods
3.2.11 Materials and reagents used for the brilliant black reduction test (BRT- MRL screening test)
- BRT MRL Screening test kits AM GMBH, Munich, Germany
3.2.11.1 Equipments used for BRT MRL screening test
- Microtiter plate with coated wells AM GMBH, Munich, Germany - Thermo block AM GMBH, Munich, Germany
3.2.12 Materials and reagents used for measuring PGE2 - PGE2 assay Kits
- Indometacin - Dimethylsulphoxide
R&D Systems, Inc., Minneapolis, USA Sigma-Aldrich, Steinheim, Germany Merck, Darmstadt, Germany
3.2.12.1 Equipments used for measuring PGE2
- Goat anti-mouse microplate - Plate covers-adhesive strips - Biopsy punch
- ULTRA-TURRAX 20000 UpM, Tp 18-10 - Polypropylene tubes
R&D Systems, Inc., Minneapolis, USA R&D Systems, Inc., Minneapolis, USA pFm, Köln, Germany
IKA WERKE, Breisgau, Germany Sarstedt AG & Co. Nümbrecht, Germany
3.2.13 Materials and reagents used for the bouillion dilution test - NaCl
3.2.14 Materials and reagents used for histology and histochemistry - Methanol
- Acetone - Ethanol
AppliChem GmbH, Darmstadt, Germany Sigma-Aldrich, Seelze, Germany Riedel-de Häen, Seelze, Germany
Materials and methods
- F(ab)2 Goat anti-mouse IgG: FITC - Mayer´s hemalum solution
Serotec GmbH, Düsseldorf, Germany Merck, Darmstadt, Germany
Sigma-Aldrich, Steinheim, Germany Leica Microsystems GmbH, Nussloch,
Germany
SAFC Supply solutions,St.Louis, USA Sigma-Aldrich, Steinheim, Germany Sigma-Aldrich, Steinheim, Germany
3.2.14.1 Equipments used for histology and histochemistry - Microme HM 560 M
- Leica microscope 020-519.511DMLB 100T
- Axiocam
- Microscope glass cover slips - Microscope slides
- Hybridization oven
- Surgical blade
Microm GmbH, Walldorf, Germany Leica Mikroskopie & Systems GmbH, Wetzlar, Germany
Zeiss, Oberkochen, Germany
Chance propper LTD, Warley, England Gerhard Menzel GmbH, Braunchweig, Germany
Gesellschaft für Labortechnik mbH, Burgwedel, Germany
Bayha, Tuttlingen, Germany
Materials and methods
3.2.15 Materials used for succinate dehydrogenase test (SDH) - KCl
SAFC Supply solutions, St.Louis, USA Sigma-Aldrich, Steinheim, Germany
3.2.16 Materials used for pyruvate kinase test (PK) - Triethanolamine
3.2.17 Materials used for isolated perfused bovine udder - NaCl
Materials and methods
3.2.17.1 Equipments used for isolated perfused udder - Water bath Mod. SWBD 5030
- Flexible pump tube 9553-75
- Surface thermometer (HI 93530) with surface sensor (HI 766B2)
- Adhesive tapes - Surgical blade - Perfusat reservoir
Stuart Scientific Co. Ltd., Exeter, Great Britain
3.2.18 Materials and reagents used for the lactate dehydrogenase assay (LDH) - LDH cytotoxicity assay kit Cayman Chemical Company, Ann Arbor,
USA
3.2.19 Materials and reagents used for the glucose assay
- Glucose assay kit Cayman Chemical Company, Ann Arbor, USA
3.2.20 Materials and reagents used for the lactate test
- Lactate test Roche GmbH, Mannheim, Germany
3.2.21 Analytical equipments
Dynatech Laboratories, Denkendorf, Germany
Eppendorf, Hamburg, Germany Heidolph, Kehlheim, Germany Stuart scientific Co, Ltd, Staffordshire, Great Britian
WTW, Weilheim, Germany
Materials and methods
- Precision Scale ALS 120-4
- Water bath 1083
- Sonication Sonorex.RK52 - Magnetomix® M1 stirrer
Labor-u.Analysen-Technik, GmbH, Garbsen, Germany
Kern & Sohn GmbH, Balingen, Germany Gesellschaft für Labortechnik , m.b.H &
CO, Hannover, Germany
Bandelin electronic, Berlin, Germany Colora Messtechnik GmbH, Lorch,
Württ, Germany
3.2.22 Solutions and buffers
PBS (0.01 mol/l; pH 7.4)
- 137 mmol/l NaCl Merck, Darmstadt, Germany - 2.7 mmol/l KCl Merck, Darmstadt, Germany - 6.5 mmol/l Na2HPO4 • 2H2O Merck, Darmstadt, Germany - 1.5 mol/l KH2PO4 Merck, Darmstadt, Germany
Block-Ag-Retrievel - 100 ml PBS
- 0.25 % BSA Sigma-Aldrich Chemie GmBH, Steinheim, Germany - 1 % Triton X 100 Sigma-Aldrich, Steinheim, Germany
Histochemistry buffer
0.2 M phosphate buffer (PH 7.4) contains - 0.2 M Na2HPO4
- 0.2 M KH2PO4 - 200 mM Succinic acid
- 1.34 mM Nitrozolium blue
Tyrode solution - 136 mmol/l NaCl - 11.9 mmol/l NaHCO3
Materials and methods
- 5.5 mmol/l Glucose • H2O - 2.7 mmol/l KCl
- 1.8 mmol/l CaCl2 • 2H2O - 1.05 mmol/l MgCl2 • 6H2O - 0.461 mmol/l NaH2PO4
SDH reaction solution - 50 mM KCl
- 10 mM Tris – HCl - 1 mM EDTA - 1 mg/ml BSA
- 1 mM KCN ; pH 7.4 - 10 mM Succinate - 6.16 mM Cytochrome C
PK reaction solution - 97.5 mM triethanolamine - 13 mM MgSO4
- 74 mM KCl - 185 µM NADH - 1 mM PEP - 2.5 U/ml LDH - 3 mM ADP
Neutral red solution 1 (1 % CaCl2, 0.5 % formaldehyde) - 6.5 ml Formaldehyde
- 50 ml CaCl2
- 445 ml Aqua bidest.
Neutral red solution 2 (1 % acetic acid, 50 % ethanol) - 4.75 ml acetic acid
- 250 ml ethanol (95 %) - 245 ml Aqua bidest.
Materials and methods
Wash Buffer (IL-1beta, TNF-alpha, IL- 6)
IL-1beta TNF-alpha IL-6
Tween 20 0.250 ml 0.250 ml 0.250 ml
PBS 500 ml 500 ml 500 ml
Reagent Diluent (IL- 1beta, IL- 6)
TNF-alpha IL-6
BSA 0.8 g 0.8 g
PBS 80 ml 80 ml
Reagent Diluent (IL-1beta) - 0.05 g BSA
- 0.438 g NaCl - 0.121 g Tris HCl - 25 µl Tween 20
Added in 50 ml Aqua bidest.
Blocking Buffer (IL-1beta) - 0.3 g BSA
- 0,015 g NaN3 Added in 30 ml PBS
Eosin solution
2.5 g eosin in 250 ml Aqua bidest. + 3 drops of acetic acid
Dendritic cell medium (DC-medium) - ß- Mercaptoethanol
- RPMI supplemented with FCS + penicillin/streptomycin
Materials and methods
3.3 Methods 3.3.1 Bovine udders
All bovine udders (used for mammary epithelium and mammary fibroblast cell cultures) were obtained from recently slaughtered cows. The first study was performed to establish a protocol to culture mammary epithelium cells. Later, when contamination of fibroblasts appeared, the second protocol was established to separate mammary epithelium cells from mammary fibroblasts. The bovine udders were obtained immediately after slaughtering, at different ages from the slaughterhouse in Minden-Lübbecke, Germany. The udders were transported to the laboratory within 75 minutes. All the experiments of the udder nearly started within half an hour after receiving the udder.
3.3.2 Cell culture experiments
Murine fibroblast (L929) cells: They are monolayer fibroblast cells obtained from male mice at the age of 100 days and were obtained from connective tissue, subcutaneous, areolar and adipose tissue.
Raw murine macrophages: They are monocytes/macrophages from adult male mice with ascites. During passaging, no trypsin or EDTA was added to detach the cells. They were detached using cell scrapers.
Primary murine macrophages: Primary murine macrophages originated from bone marrow of the mice femur; therefore, the bone marrow was collected from the mice femur containing dendritic cells and primary macrophages. After incubation in DC medium, primary macrophages were attached to the bottom of the petri dish and were thereby separated from the dendritic cells.
3.3.2.1 Primary mammary epithelium cell cultures
Mammary epithelium cells were cultured by leaving the cells to grow out of the explants. Bacterial and fungal contamination initially appears after culturing the explants. For this reason, a new protocol was established using collagen as a coating for 6-well plates in order to have best results in terms of differentiation when using collagen gels which allow the cells to be confluent. When the collagen released from
Materials and methods
the bottom of the well, cells shrink which in other terms stimulate the cells to differentiate (EMERMAN and PITELKA, 1977).
Washing process was done as follow:
Tissues taken from teats and mammary glands were cut in small pieces (1 cm3); the tissues were incubated in Braunol (1:10 dilution in PBS) for 15 minutes, they were washed in sterile PBS up to 5 times, then they were washed in DMEM-F12 supplemented with 20 % FCS, 10 % amphotericin B, 10 % gentamicin and 10 % penicillin/streptomycin. Afterwards, they were placed on an orbital shaker for 10 min, washed again in sterile PBS up to 5 times, and were washed with DMEM-F12 supplemented with 10 % FCS, 1 %, amphotericin B, 1 % gentamicin and 1 % penicillin/streptomycin.
According to HU et al. (2009) culturing was done with some modifications:
The tissues were incubated in DMEM-F12 supplemented with 10 % FCS, 1 %, amphotericin B, 1 % gentamicin and 1 % penicillin/streptomycin and protease 0.6 mg/ml in a petri dish over night in a fridge; the tissues were taken out to a new petri dish containing DMEM-F12 with 10 % FCS, 1 %, amphotericin B, 1 % gentamicin and 1 % penicillin/streptomycin. The tissues were agitated gently and were incubated at 37°C, 5 % CO2 for one hour to attach fibroblasts and detach epithelium cells in the supernatants.The supernatants contained the detached epithelial cells were collected, and then the collected supernatants were centrifuged at 600 x g for 5 min.
The medium was removed, fresh medium was added, and they were centrifuged again. Afterwards, they were washed 3 times, and then 5 ml of DMEM-F12 supplemented with 10 % FCS, 10 % penicillin/streptomycin , 10 % gentamicin, 10 % amphotericin B, 25 µg/ml EGF, 10 % L-Glutamine and 5 ml Insulin-transferrin-Selenium A were added. Afterwards, 6-well plates were coated with rat tail collagen (2 mg/ml) by spreading 33 µl per well using a sterile cell scraper. They were left to dry under the sterile bench for one hour. Afterwards, the plates were flushed with sterile PBS. Afterwards, the cells were counted and seeded in 6-well plates at a density of about 500.000 to 1000.000 cells per well. Later, the medium was changed after 48 hours, then every 24 hours after washing with sterile PBS. Cells were confluent after 8-10 days and were passaged into 25 cm² culture flasks at a density
Materials and methods
of 1000.000 - 2000.000 cells/well. Cells were detached by adding 1 % EDTA solution for 5 min at 37°C and 5 % CO2, then 0.05 % trypsin/0.02 % EDTA solution for 10 min at 37°C and 5 % CO2 until cells were detached and separated followed by adding DMEM-F12 with 10 % FCS to inactivate the trypsin. The cells were centrifuged for 10 min at 600 x g at 4 °C, and seeded at a density of 10.000-20.000 cells/well using a 96-well plate.
For the experiments, primary mammary epithelium cells were used at passages 4-7.
3.3.3 Separation of primary mammary epithelium cells contaminated with mammary fibroblasts
In the case of using trypsin digestion, epithelial cells were isolated after 3 passages.
The degree of digestion and culture conditions seemed to be important for cell development. For detaching epithelial cells from culture dishes or culture flasks, it took 10 to 15 minutes, while the fibroblasts needed less than 1 to 2 minutes, but this method only succeeded for one or two passages (HU et al., 2009).
According to FRESHNEY and FRESHENEY (2002), primary mammary epithelium cells were separated from primary mammary fibroblasts as follow:
The cell mixture was seeded into a flask for 30 min to 1 hour. Then the mixture was transferred into another new flask which was done for intervals of 1-3 hours up to 24-48 hours. Commonly the fibroblasts tend to attach first at the bottom of the flask, while the epithelial cells remain in the suspension and attach in the later seeded flask.
3.3.4 Culturing primary mammary fibroblasts
Tissues taken from the teats and mammary glands were sterilized and washed as mentioned in 3.3.2.2, then culturing followed according to HU et al. (2009):
the tissue explants were cut into small pieces about 1 cm3 in 6-well plates, and 1-1.5 ml DMEM-F12 supplemented with 10 % FCS, 10 % penicillin/streptomycin, 10%
gentamicin, 10 % amphotericin B, 25 µg/ml EGF, 10 % L-Glutamine, 5 ml Insulin-transferrin-Selenium A was added. The explants were incubated at 37°C and 5 % CO2 for 45-60 min, monitored every 30 min. If the adjacent area surrounding the tissue was dry, several drops of medium were added ensuring the tissue would not
Materials and methods
float and separate from the bottom of the culture dish. After 4 hours, 0.5 ml medium was added to every culture dish and 1 ml medium was added every 48 hours until cells were visibly spread across the bottom of the culture dish. When the cells became confluent after 10-15 days, they were passaged into 25 cm² culture flasks at a mean density of 1000.000 to 2000.000 cells/well, using 1 % EDTA for 1 min, and 0.25 % trypsin, 0.02 % EDTA for 3 min, 37°C and 5 % CO2. DMEM-F12 containing 10 % FCS was added to inactivate the trypsin, then centrifugation took place for 10 min at 2357 x g at 4°C. Finally, the cells were seeded in 96-well plates at a mean density of 10.000-20.000 cells/well in fresh whole medium.
For the experiments, primary mammary fibroblast cells were used at passages 5 to 8.
3.3.5 Cell counting
To determine the cell count, cells were stained with trypan blue and counted using a Neubauer chamber: 50 μl of a cell suspension were mixed with 100 μl trypan blue suspensions (40 mg trypan blue in 10 ml Aqua bidest.) and 10 μl of this suspension was placed in the counting chamber.
The cell count was determined in four quadrants with a 100-fold magnification under the light microscope. Trypan blue enables a differentiation of viable and dead cells.
All cells incorporate the stain, but only viable cells are able to eliminate the blue stain.
When viewed under the microscope, dead cells have a blue cytoplasm and nucleus, whereas viable cells are not stained. The total cell count can be calculated with the following formula (1):
Formula (1): calculation of total cell count.
Number of counted cells x dilution x 5 x 104
= cell absolute
Number of counted squares
Cells absolute = absolute cell count in 1 ml of culture medium Number of counted cells = number of counted cells in 4 quadrants 5 = the cell were suspended in 5 ml medium
104 = cells under the glass slide
Materials and methods
3.3.6 Immunocytochemistry
Primary mammary epithelium and primary mammary fibroblasts were verified by indirect immunofluorescence staining. The monoclonal mouse anti-vimentin antibody was used in a 1:100 dilution, the monoclonal mouse anti-cytokeratin antibody was used in a 1:300 dilution, and both were diluted in blocking-Ag-retrieval solution. The secondary antibody (FITC) was used in 1:200 dilution and bisbenzimid was used in a 1:100 dilution in Aqua bidest. The staining protocol was done as follow:
Cells were grown in 6-well plates on sterile cover slips up to 70 % confluence; they were washed twice with PBS and fixed with cold methanol/acetone (50/50; v/v) for 5 min.. Afterwards, the slides were either stored in sterile PBS at 4°C until further processing or stained immediately. The slides were washed with PBS (5 min, max. 2 times), and were incubate with blocking-Ag-retrieval solution for 30 minutes at room temperature. Afterwards, they were incubated with the primary antibody (anti-vimentin & anti-cytokeratin) for 1 h at 37°C, then they were washed with PBS (5 min, max. 2 times), and were incubated with the fluorescence-labelled secondary antibody (FITC) for 30 min at 37°C in the dark. After washing with PBS (5 min, max. 2 times), the slides were stained with bisbenzimid at room temperature for 15 min in the dark, and were washed again with PBS for 5 min.
100 µl of the antibody and bisbenzimid solutions were added per slide and 1 ml of blocking-Ag-retrieval solution were added per slide for the washing process. During the entire process of the staining, drying of the wells and slides was strictly avoided.
3.3.7 Treating of the cells 3.3.7.1 AgNO3treatment
A stock solution of 1 mmol/l AgNO3 was prepared and different concentrations of AgNO3 (control (medium), 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 mmol/l) in different media or NaCl solution according to the cell type were prepared;
the cells were treated and were incubated at 37°C and 5 % CO2 for 24 h. Afterwards the supernatants of the cell incubation experiments were collected and frozen -20°C for measurement of biomarkers of inflammatory reactions by ELISA. The samples were in 7 duplicate numbers per dilution.
Materials and methods
3.3.7.2 Incubation with MgAg1% sticks
MgAg1% sticks were sterilized and incubated in different types of media according to the cell type or NaCl with different volumes (1, 3 and 10 ml), in addition to one tube of medium as negative control; they were incubated in a water bath at 37°C and shaken for 5 days. Afterwards, the supernatants were taken to treat the different cell types, which were incubated at 37°C and 5 % CO2 for 24 h. The supernatants of the cell incubation experiments were collected and frozen at -20°C for biomarkers of inflammatory reactions by ELISA. The samples were in 7 duplicate numbers per dilution.
Materials and methods
3.3.8 Degradation process of MgAg1% sticks 3.3.8.1 The isolated perfused udder
3.3.8.1.1 Preparation of the udder
Within 10-15 min post-slaughter, the udders were transported over 45 min in a plastic sacks to the laboratory. Blood clots in the vessels of the glands were cleared using 1 l heparinised (1 %) tyrode solution for each half udder. The udder was fixed in a
‘‘natural’’ position to a metal frame. Within a few minutes, the large arteries of the udder (the right and left A. pud. ext) were supplied with the perfusion fluid delivered via silicone tubes with suitable diameter using a peristaltic pump, while the larger veins (cranial and caudal superficial epigastric vein) were cannulated to allow sampling and removal of the perfusate (Fig. 6). Smaller veins were closed using artery forceps (KIETZMANN et al., 1993; EHINGER and KIETZMANN, 2000 a, 2000 b).
3.3.11.1.2 Starting the perfusation and controlling the viability
To inhibit the development of tissue odema, each udder half was perfused with tyrode solution (Fig. 7). To ensure a perfusate flux of 100-120 ml/ min, the pressure was 50 mm Hg. The mammary glands were milked during 30 min equilibration phase.
The viability of the perfused udder was controlled using biochemical parameters such as lactate dehydrogenase activity (LDH), glucose consumption and lactate production in the perfusate (KIETZMANN et al., 1993; EHINGER and KIETZMANN,
Fig. 6: The mammary vein and mammary artery are shown here (A) on the carcass of a cow after the udder has been cut away (B). The isolated perfused udder after preparation.
B
A
Materials and methods
2000 a, 2000 b). The udder skin temperature was measured every 60 minutes.
Udders which exceeded the limits were not included in the test evaluation.
Fig. 7: Schematic design for isolated perfused udder (EHINGER, 1998)
3.3.8.1.2 Measuring parameters of viability of bovine udder
Lactate dehydrogenase enzyme (LDH) is a soluble enzyme located in the cytosol.
LDH can be used as an indicator of cell membrane integrity and is also a measurement of cytotoxicity. The assay was performed according to the protocol of the manufacturer as follows:
100 µl of the perfusate from each udder were pipetted in 96-well plate. They were centrifuged at 400 x g for 5 min. 100 µl of the standards were prepared as mentioned in the manufactures protocol, then 100 µl of each supernatant from each well was transferred to the new plate, 100 µl of reaction solution was added to each well.
Afterwards, the plate was incubated on an orbital shaker for 30 min. at room temperature. The measurements were performed using photometer with a wavelength of 490 nm.
Glucose concentration: It is the most important carbohydrate in biology transported through blood stream. It is the primary source of energy for the body cells. The assay was applied according to the protocol of the manufacturer as follows:
Materials and methods
5 µl of each perfusate sample was added in tubes, 500 µl of the enzyme mixture was added and then mixed well. The tubes were incubated at 37 °C for 10 min., and finally 150 µl was taken away from each tube to a 96-well plate. The measurements were performed using photometer with a wavelength of 520 nm.
Lactate concentrations: Anaerobic glycolysis increases blood lactate and pyruvate levels. The test was done as follows:
A standard calibration curve was prepared from 5 mg/ml tyrode with concentrations of zero, 0.1, 0.3, 0.6, 0.9, 2 and 1 mg/ml lactate, 833 µl of reagent 1 was mixed with 167 µl of reagent 2, then 2 µl of each standard was added per well. 2 µl of the perfusate was added per well. 100 µl of the reaction medium was added per well, the plate was incubated for 10 min in room temperature. The measurements were performed using photometer with a wavelength of 520 nm.
3.3.8.2 The degradation experiments
The bovine udder was perfused with tyrode solution, sterile MgAg1% sticks (52-56 mg) were administrated intracisternally and left in the teat cistern for 5-6 hours, while another teat was used as a control. The teats were cut and frozen at -20°C for
The bovine udder was perfused with tyrode solution, sterile MgAg1% sticks (52-56 mg) were administrated intracisternally and left in the teat cistern for 5-6 hours, while another teat was used as a control. The teats were cut and frozen at -20°C for