Appendix 1 SOLUTIONS FOR MIGRATION STUDIES 1.1 Phosphate Buffered Saline (PBS) (0.145 M NaCl, 0.01 M Na2PO4, pH 7.1) 8.5 g NaCl BDH 1.07 g Na2HPO4 (anhydrous) BDH 0.39 g NaH2PO4.2H2O M&B Ltd. The salt mixture was made up to 1 L with double distilled water, and stored at 4oC. 1.2 PBS / 2%BSA / 01.%NaN3 10 g Bovine serum albumin CSL 0.5 g NaN3 BDH The BSA and NaN3 was gently floated onto 500 mL of PBS and allowed to dissolve, without stirring or mixing. The solution was stored at 4oC. 1.3 FACS Fixative 13 mL Formalin (40% Formaldehyde) M & B 10 g Glucose BDH 1.09 mL 15% NaN3 BDH The above were dissolved in 500 mL of PBS and the resultant solution was stored at 4oC. Appendix 2 Solutions for Cell Culture 2.1 Supplemented RPMI 1640 The culture medium RPMI 1640 (Cytosystems) was reconstituted from powder with Milli Q purified water and sterilised by 0.2 mm vacuum filtration. The following supplements were added to the medium immediately prior to use. 5 % (w/v) Foetal Calf Serum CSL 0.3 % (w/v) L-Glutamine Sigma 0.1 % (w/v) 2-Mercaptoethanol Sigma 100 U / mL Benzylpenicillin CSL 2.2 Trypsin The 0.05% trypsin solution was prepared as follows from the following 3 stock solutions. Solution 1 Trypsin diluent 8.0 g NaCl BDH 0.4 g KCl BDH 0.06 g Na2HPO4 BDH 0.06 KH2PO4 Ajax Chemicals Ltd The salt mixture was made up to 1 L with distilled water, autoclaved and stored at 4oC. The solution has a known stability of approximately 6 months. Solution 2 EDTA 2 % (w/v) pH 7.3 2.5474 g EDTA disodium salt SERVA (Ethylenediaminetetraacetic acid) The salt was made up to 100 mL with distilled water and the pH was adjusted to 7.3 using 1 N NaOH. The solution was autoclaved, stored at 4oC and has indefinite stability. Solution 3 Trypsin Stock Solution 2.5 % 25 g Trypsin Boehringer Mannheim 8.0 g NaCl BDH 0.4 g KCl BDH 0.06 g Na2HPO4 BDH 0.06 g KH2PO4 Ajax Chemicals Ltd 50 g Glucose BDH The above were made up to 1 L with double distilled water and stored at -20oC. For the active 0.05% Trypsin, solutions 1, 2 and 3 were combined as follows: Solution 1 48.5 mL Solution 2 0.5 mL Solution 3 1.0 mL 2.3 Cell Counting Procedure The concentration of the cell suspension was determined as follows. A small aliquot (approximately 50 mL) of the cell suspension was removed and placed under the coverslip on a Neubauer Haemocytometer counting chamber. The number of viable cells in 5 small squares on the grid were counted (minimum of 100). The following formula was used to calculate the cell concentration. cell concentration = number of cells X 1 x 105 (cells/mL) 2 If the density of the cell suspension was low, 4 of the large squares were counted and the above formula was divided by 20. 2.4 Crystal Violet Assay for Cell Viability At the end of the incubation period the adherent cells were stained as follows. The culture media was flicked from the wells and the cells were washed with 100 mL of PBS. The PBS was then flicked from the wells and the cells were fixed with 100 mL of methanol (BDH) for approximately 2 minutes. The methanol was then flicked from the wells and the plate was allowed to air dry for approximately 5 minutes. The adherent cells were then stained with 100 mL of aqueous crystal violet (1% w/v) (RAL Reactifs) for 5 minutes. The excess stain was then washed out by immersion of the plate in a beaker of clean water. The wells were flicked dry and the cells were solubilized with 100 mL of 33.3 % acetic acid (Ajax). 2.5 Settings for the ELISA plate reader The following settings were used for the ELISA plate reader (Titertek Multiskan - ICN) when reading TNFa Bioassay plates. Dual beam Mode 2 Reference Wavelength (lm) 1st filter = 2 (lm = 414 nm) Test Wavelength (lm) 2nd filter = 6 (lm = 520 nm) Appendix 3 Solutions for RNA Extraction 3.1 4 M Guanidinium Thiocyanate (Solution D) The following were dissolved in 293 mL of water by heating at 65oC. 162 g Guanidinium Thiocyanate Fluka 17.6 mL of 0.75 M NaCitrate, pH 7.0 M & B 26.4 mL of 10% Sarcosyl Sigma The solution was filtered through a 0.45 mm Milliporeª filter, and 2-Mercaptoethanol was added to a final concentration of 0.75%. (N.B. Do not autoclave) 3.2 TE Saturated Phenol Phenol (ICN Biomedicals) was melted in a water bath at 65oC. TE (Tris buffered ethylenediaminetetraacetic acid) buffer was added to the melted phenol to a concentration of 40 % (w/v), the mixture was shaken for 5 minutes and then placed at 4oC overnight to allow the phases to separate. The upper aqueous phase was then aspirated and fresh TE buffer was added to a final concentration of 25 % (w/v). The mixture was shaken for 5 minutes and then rested at room temperature for 2 hours. The aqueous phase was aspirated and 8-Hydroxyquinoline (MERCK) was added to 0.1% (w/v). The TE saturated phenol was stored at 4oC in dark container. Appendix 4 Solutions for RNA Analysis 4.1 1.2% (w/v) Formaldehyde Agarose Gel The following were heated in a microwave at high power setting for 2.5 minutes. 1.2 g HGT agarose ICN Biomedicals Inc. 20 mL 5 x MOPS buffer BDH Chemicals Ltd. 62.1 mL Milli Q purified water The agarose mixture was allowed to cool to approximately 60oC before adding 17.9 mL of formalin (40% formaldehyde), following which the gel was poured into a horizontal slab gel plate with the appropriate well combs inserted. After 1 hour the combs and gel plate ends were removed and the gel was ready for use. 4.2 10 X MOPS Buffer (0.4 M MOPS, 100 mM NaAc, 10 mM EDTA, pH 8.0) 83.72 g of MOPS BDH 33.34 mL of 3 M Sodium Acetate Ajax Chemicals Ltd 10 mL of 1 M EDTA, pH 8.0 SERVA The mixture was made up to 1 litre with double distilled water, buffered with 5 M NaOH to pH 7.0 and autoclaved. 4.3 RNA Loading Buffer 50% Glycerol M & B 1 mM EDTA SERVA 0.4% Bromophenol blue M & B 4.4 20 x Sodium chloride, Sodium Citrate (SSC) (3 M NaCl, 0.3 M NaCitrate) The following were added to 800 mL of Milli Q purified water. 175.3 g NaCl BDH 88.2 g NaCitrate M&B The solution was made up to 1 L and the pH was adjusted to 7.0 with HCl. 4.5 TE Buffer (10mM Tris HCl pH 7.6, 1 mM EDTA) The following were added to 800 mL of Milli Q purified water 1.58 g Tris HCl Sigma 0.38 g EDTA SERVA The solution was made up to 1 L with Milli Q purified water, buffered to pH 7.4 and autoclaved 4.6 cDNA Hybridisation Buffer Notes 1 Formamide must be a solid at -20oC or else it is not deionised. 2 Blotto must be completely dissolved in formamide before the SDS is added (heating at 65oC and vigorous shaking may required). 3 Heating the 50% Dextran Sulfate in a water bath at 65oC for approximately 15 minutes prior to use makes it easier to pour. 4 The buffer may be stored at 4oC for approximately two weeks prior to use. The following were mixed in order. Amount Final per 50 mL Conc. Formamide (100%) 25 mL 50% (MERCK) Blotto (Milk powder) 0.25 g 0.5% (Nestle, Australia Ltd) 10% SDS 5 mL 1.0% (Sigma) 50% Dextran sulfate 10 mL 10% (Pharmacia) 2 M Phosphate buffer pH 6.4 1.25 mL 50 mM (KH2PO4 & Na2HPO4) 20 x SSC 10 mL 5 x SSC Immediately prior to use add 2.5 mL of 10 mg/mL (final concentration of 0.5 mg/mL) Salmon Sperm DNA (ssDNA) (Boeringher Mannheim). The ssDNA was denatured by heating at 95oC for 5 minutes and then cooled on ice prior to addition to the hybridisation buffer.