Chapter Three

Materials and Methods

3-1 Introduction
The biological medium in which the chemical reactions occur is a weakly conducting electrolyte, this makes it difficult to establish a strong electric field. External electric fields, therefore, seem to be restricted. These restriction doses not apply to external magnetic field because the medium is essentially nonmagnetizable. The applied magnetic field therefore is not affected by the continuum encountered in biological system [1].

3-2 Magnetic System

For a given magnetic system, magnetic field intensity, field uniformity and air gap width are closely related and strongly influenced by the choice of pole caps. When specifying an electromagnet with pole caps of cylindrical or conical tapered design: 1- Field intensity varies directly with the pole piece diameter of the magnet, the power input and inversely as the air gap width and pole cap face diameter. 2- Field uniformity varies directly with pole cap face diameter, therefore with the pole diameter of the magnet, and inversely as the field intensity and air gap width [2].

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The magnetic system used in this research is shown in Fig.(3-1,3-1a), which was constructed by Varian (V2901 Regulated Magnet Supply associates).Avery important advantage of electromagnets is the degree of control, which can be exercised over the magnet system. The field can continuously be adjusted to the desired value by controlling the amount of current delivered to the magnet coils. Generally, the controls are an integral part of power supply furnished as part of a magnet system. A power supply is generally chosen which provides a DC output that maximizes the field producing capabilities of the electromagnet. Since the major portion of the power is converted to heat in the magnet coils, these too require watercooling. The magnetic system was cooled using pump motor for circulating water in the cooling system of the magnetic system to circulate cooling water with high flow.

0100090000035700000001001c000000000004000000030108000500 00000b0200000000050000000c02e90dad0e040000002e0118000400 000002010100040000002e011a001c000000fb029cff0000000000009 001000000000440001254696d6573204e657720526f6d616e0000000 000000000000000000000000000040000002d0100000d000000320a5 a00790e0100040000000000a60ee40d20572d00040000002e0118000 30000000000

Figure (3-1) Picture of the magnetic system(side)

0100090000035700000001001c000000000004000000030108000500 00000b0200000000050000000c02e90dad0e040000002e0118000400
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000002010100040000002e011a001c000000fb029cff0000000000009 001000000000440001254696d6573204e657720526f6d616e0000000 000000000000000000000000000040000002d0100000d000000320a5 a00790e0100040000000000a60ee40d20572d00040000002e0118000 30000000000

Figure (3-1a) Picture of the magnetic system (front)

3-2-1 Calibration of the Magnetic System

The calibration of magnetic system was done by magnetometer measuring instrument (DC magnetometer, USA) the principle of operation as the core saturated equally in both directions; signal generated when unknown field unbalances core. The calibration curve shown in Fig.(3-2).This magnetometer consists of a probe connected to a DC meter, it has a measuring range of 1mG-20kG, with accuracy 0.001%.The calibration curve was obtained at three different air gap width (7cm, 9cm, 12cm) to get different magnetic field intensities.

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5000 4500

(x10- 4 )( T ) Magnetic Field

4000 3500 3000 2500 2000 1500 1000 500 0 0 4

d= 7cm d= 9cm d= 12cm (d = 7 c m ) L in e a r D C C u rre n t (A ) (d = 9 c m ) F ig u(3e- 2:C o rre la tio n b e tw e e n (A)C ncde a r n t r ) D L in u rre a m a g n e tic (T e s )fo r d iffe re n t a ir(d = p s2 c m ) fie ldla ga 1 L r 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8in5e2a 5 6 6 0

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3-2-2 Magnetic Field Exposure

Exposure to magnetic field is performed using DC-electromagnet, which was constructed by Varian with maximum air gap width 12 cm, and pole cap diameter 5cm, which provided a highly homogenous field. By changing the DC-current, the magnetic field can be changed. As our studies require the maintenance of constant sample temperature 37C (human body temperature) over relatively long periods of time. A magnet system with well designed efficient cooling circuits facilitates sample temperature control since, a bout 95%of the input power is discharged as heat through the water, add a modest room air conditioner can easily handle the reast.Tube of sample was exposed to magnetic field in a small water bath, at 37C 0.2C , which placed between magnetic poles. An identical water bath with control sample was placed in the natural static magnetic field SMF (0.5G). Both baths were constructed at engineering workshop at Al-Nahrain University,it was designed in such way that the water enters the two bath at same time and coupled with a mean water bath with a thermostat to form a closed system of water circulation and temperature control in as shown in Figs.(3-1,3-3).

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(4)helmoholtze coil, (5)magnetic poles, (6)magnetic fluxmeter, (7) current adjustment

0100090000035700000001001c00000000000400000003010800050000000b0 200000000050000000c02e90dad0e040000002e011800040000000201010004 0000002e011a001c000000fb029cff0000000000009001000000000440001254 696d6573204e657720526f6d616e00000000000000000000000000000000000 40000002d0100000d000000320a5a00790e0100040000000000a60ee40d2057 2d00040000002e011800030000000000

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3-3 Biological Work 3-3-1 Materials Used in the Present Work

Apparatus Water bath Analytical balance Incubator Autoclave Centrifuge Hotplate-magnetic stirrer Refrigerator Distillation apparatus Light microscope a- Apparatus The Apparatus which are used in the present work .are shown in table (3-1) Table (3-1): The Apparatus used Manufactured Company Gallen Kamp/Germany Sartorious/Germany Sanyo /Japan Tommy/Japan BHG/Germany Rod well/England Ishtar Exdo W.OFIAIG and sons Olympus

b- Tools The tools which are used in the present work are shown in table (3-2) Table (3-2): The Tools used

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Tools Pasteur pipette Micropipette Screw capped bottles Sterile vacutainer siliconized tubes Milipore filters Slides Haemocytometer Glass ware c- Chemicals

Manufactured Company Biomerious Orgamon Netheler Terumo Sherwood Whatman Blue stare

The chemicals which are used in the present work are shown in table (3-3) Table (3-3): The chemicals used Chemicals KCl Methanol Ethanol Glacial Acetic Acid Giemsa Stain RPMI-1640 NaCl Na2HPO4 KH2PO4 Sodium bicarbonate NaHCO3 Hepes buffer Crystallin penicillin Sterptomycin sulphate Phytohaemagglutinine(PHA) Staphylococcus aureus Trypan blue Nutrient Agar Manufactured Company BDH/England BDH/England BDH-pool/England BDH/England BDH-pool/England Sigma/USA Sigma/USA BDH/England BDH/England Sigma/USA Flow laboratories Denmarke France Iraqi center for cancer Researches Al-Nahrain University/Biotechnology Dep. Al-Nahrain University/Biotechnology Dep. Al-Nahrain University/Biotechnology Dep.

3-3-2 The Preparation of Samples

The experiment was performed on three normal or healthy human 47

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bloods. Blood samples (5 milliliters) were collected into heparinised sterile tubes and were processed within one hour following sample collection.150 samples were collected.

3-3-3 Preparation of Solutions, Buffer and Stock Solution Used in Lymphocyte Transformation Assay
Lymphocyte transformation assay was used to assess the lymphocyte responses and cell mediated immune response for healthy people, which occur when exposed to several magnetic field intensity by exposed to different times of exposure. a- Solutions a-1 Antibiotic Solution Stock solution of antibodies was prepared by dissolving 1000000 IU of crystalline penicillin and 1g streptomycin sulphate in 100ml distilled water, sterilized by 0.22mm millipore filter and stored at -20C. a-2 Phosphate buffer saline (PBS) solution This buffer was prepared by dissolving 8g of NaCl, 0.2g of KCl, 1.15g of Na2HPO4, 0.2g of KH2PO4 in 100ml distilled water the PH of this solution was adjusted to 7.2. 48

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a-3 Fixation solution It was prepared freshly by mixing 3 volumes of absolute methanol and 1 volume glacial acetic acid in the ratio of (3:1v/v).

a-4 Sorensons buffer solution This solution was prepared by dissolving 1.804g of Na2HPO4 and 1.8136g of KH2PO4 in 200ml distilled water, adjusted the PH to 6.8.The stock solution was stored at 4C. a-5 Hepes(N-2-hydroxyelthyl piperazine-N-2-ethane Sulphonic acid) solution By using hepes buffer supplied from Flow Company to modify PH in ratio 100:1 ml from media and the PH should be between 7.5-7.4 prepared with concentration of 238.5mg/ml. a-6 Hypotonic solution It was prepared by dissolving 0.5587g from KCl powder in 100ml of distilled water, with concentration (0.075M) the stock solution was stored at 4C. a-7 Serum (Sigma Chemical Company)

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Fetal calf serum (FCS) was inactivated by heating at 56C for 30min., then dispensed into 20ml aliquots and stored at 20C a-8 Sodium bicarbonate Sodium bicarbonate NaHCO3 (Analar) 4.4g, 100ml D.D.W (double distillated water) sterilized by autoclaving, and stored at 4C. a-9 Phytohaemagglutinine (PHA) Crude PHA was obtained from Baghdad Center for Cancer Researches. Dispensed into 2ml aliquots and stored at 20C a-10 Tissue culture Media Tissue culture media (PH=7.2) was prepared by dissolving 10.4g RPMI-1640,10ml from of 1M hepes buffer with 2mg of sodium bicarbonate (Analar),10ml antibiotics,100ml serum in 900ml D.D.W, all these components was mixed well, sterilized by filtration through 0.22m millipore filter, adjusted to PH=7.2 then dispensed to 20ml aliquots and stored at 20C. b- Method of Lymphocyte Transformation Assay This assay was done according to the method described by Further et al. [71]. b-1 Blood Culturing Assay 1: 2.5 ml of RPMI-1640 tissue culture media was added to sterile tube containing 0.3 ml of heparinized blood. The tube was exposed to different magnetic field intensities (0.1-0.5T) for different times of exposure 50

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(30, 60, 90, 120 min.) to each intensity. Another tube containing similar amount of blood was used as a non exposed (control) Assay 2: 0.3ml mitogens (PHA) was dissolved in 2.5 ml of RPMI-1640 tissue culture media .Sterilised by 0.22m millipore filter, was added to sterile tube containing 0.3 ml of heparinized blood .The tube was exposed to 0.2 Tesla magnetic field for (30, 60, 90, 120 min). Another tube with PHA containing similar amount of blood was used as non exposed (control). b-2 Harvesting After 3 days of incubation at37C, the cell suspension was mixed gently, then centrifuged at 2000 rpm. for 10 minutes at room temperature, then the supernatant was discarded ,the remaining cell pellet was resuspended in hypotonic solution. b-3 Hypotonic solution The cells were resuspended in 2ml prewarmed 0.075M KCL at 37C with continuous shaking, then the volume was made up to 8ml by adding more prewarmed 0.075M KCL gradually with constant shaking. The cell suspension was then incubated at 37C for 90 minutes with accessional shaking. Cells were collected by centrifugation at 2000 rpm for 10 minutes, the supernatant was discarded and cell pellet was treated with fixative. b-4 Fixation 0.5ml of freshly made fixative (methanol and glycial acetic acid) 51

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(3:1v/v) was added drop wise with continuous agitation to the cells. Cell suspension was then centrifuged at 2000rpm. for 10 minutes at room temperature. The fixative was decanted and another 0.5ml fixative was added and the cells were collected by centrifugation. Fixative was changed 3 times before spreading the cells on the slides. b-5 Slide making This was performed by droppings 2-3 drops of cell suspension with pasture pipette,from 30 cm onto clean slid and allow to air to dry at room temperature for staining , the remaining cell suspension were stored at -20C. b-6 Staining Slides were stained with Giemsa stain, which is freshly made (1 part of Giemsa stain to 4 part of Sorenson's buffer) for 5-15 min. Slides where washed with the same buffer and left it to dry at room temperature. The number of lymphoblast and lymphocyte cells were calculated and examined under light microscope using oil immersions lens, the percentage was calculated from the following formula: % Lymphocyte transformation = lymphoblast cells / total no.( lymphoblast + lymphocyte) cells100

3-3-4 Phagocytosis Assay

a- Material

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Bacterial suspension preparation by Staphylococcus aureus obtained from Al-Nahrain University/ Biotechnology Department .It was cultured on nutrient agar media, incubated for 24 h. at 37C, the colonies was collected in PBS. The bacterial cells were then washed three times and reconstituted at concentration of 1106 cells/ml and stored at 4C.

b- Method
This method was performed according to Further et al.[71] as follows: 1ml of blood in a heparinzed tube was mixed with 1ml of bacterial suspension (1106 cells/ml),was kept as a control another one was exposed to four magnetic field intensity (0.1, 0.2, 0.3, 0.4 T).Time of exposure for each magnetic field intensity was (30 , 60, 120, 180 min.).Tubes incubated at 37C for 30 min. with slow movements. A blood film was prepared on dry clean slide, the slid covered with methanol for 5-10 min. followed by staining with freshly prepared Giemsa stain for 5-10 min., then washed with buffer solution. The percentage of phagocytosis was calculated as follows: % phagocytosis = phagocytic cells / total no.(phagocytic + non phagocytic cells) 100

3-3-5 Flow Haemocytometer

Haemocytometer has many applications in immunology one of these is measuring or the separation of live cell from dead cells. 53

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3-3-5-1 Viable and Nonviable Assay (Haemocytometer Assay) 0.3 ml of heparinized blood was added to sterile tube containing 2.5 ml of RPMI-1640 tissue culture media and was kept as control the other one is exposed to magnetic field intensity 0.2T for different time of exposure (30, 60, 90, 120 min.).By dropping one drops of cell suspension with pasture pipette, onto clean slid, and for staining, a drop from trypan blue dye on a drop of cell suspension, leave it for 3 minute then with clean pasture pipette insert the cell suspension stained with trypan blue dye between the cytometer slashes covered with slid cover. Live cell will be crystalline, dead cells take up dye color within a few second, percentage of dead cell will be calculated as shown: Dead cell % =dead cell/total number (live cell + dead cell) 100

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