a) Physiological saline
A sterile solution of 0.9 % NaCl dissolved in 100 ml of DDW.
b) Phosphate buffered saline (TBS) pH 7.4
PBS is a 0.02 M phosphate buffer containing 8 g NaCl, 1.2 g Na2H P 04.2H20 and 0.39 g NaH2P 0 4.2H20 dissolved in 1 litre of DDW.
c) Phosphate buffered saline - Dulbecco’s formula
Dulbecco’s formula PBS lOx concentrate with calcium and magnesium (Flow Laboratories Australasia Pty Ltd, North Ryde, NSW) was diluted 1 to 9 with sterile DDW.
d) Hanks balanced salt solution fHBSSl
One litre of solution contained 8.0 g NaCl, 0.4 g KC1, 0.1 g MgS04.7H20 , 0.1 g MgCl2.6H20 , 0.14 g CaCl2, 0.06 g Na2H P 0 4, 0.06 g KH2P 0 4, 1.0 g glucose , and 20 mg phenol red in DDW. The solution was autoclaved for 20 minutes at 112 °C. Before use the pH was adjusted to 7.4 (red colour) by dropwise addition of 5 % NaHC02 solution. If required, foetal calf serum (FCS) was added to the HBSS before the pH was adjusted.
e) Alsever’s solution
This solution consisted of 8g sodium citrate (Na3C^H^0y.2H9 0), 4.2 g NaCl, 20.5 g glucose and 8 ml 10 % citric acid made up to a volume of one litre with DDW.
Chapter Two 37 f) Buffered ammonium chloride solution
To one litre of DDW were added 0.1 g KH2P 0 4, 8 g NH4C1 and 1 g Na3- EDTA and the pH adjusted to 7.3. The solution was sterilised by passage through a 0.2 ßm filter.
g) Eagle’s minimum essential medium (Eagles’s MEM1
Eagle’s minimum essential medium plus Earle’s salts (F15)(Gibco, Grand Island, USA) was prepared as follows: 9.8 g dry Eagle’s MEM was dissolved in 1 litre DDW containing 44 ml 5 % sodium bicarbonate. The medium was sterilised by filtration (0.2 [im) and stored at 4 °C until required. Before use, the pH was adjusted to 7.4 by gassing with 5 % CC>2-
h) White Cell Diluting Fluid
Ten milligrams of methyl violet (George T. Gurr Ltd, London, England) was dissolved in 1.5 ml glacial acetic acid (Univar, Ajax Chemicals Pty Ltd) and
100 ml DDW. The solution was filtered through Whatman’s No.l filter paper.
i) Sodium azide in PBS
A 0.1 % solution of sodium azide in PBS was prepared by diluting a 5 % sodium azide solution 1:50. The solution was used to dilute monoclonal antibodies (mAb) and to wash cell suspensions after labelling with mAb.
j) "FACS" cell suspension medium
Cells to be analysed on the FACS, after labelling with mAb, were resuspended in a solution of F15 (190 ml) with 0.1 % sodium azide (4 ml 5 % sodium azide) and 3 % FCS (6 ml).
k) Slide Gelatin Coating Solution
To 250 ml DDW was added 0.625 g swine gelatin (Sigma Chemical Co.) and 0.063 g chrome alum (Chromium potassium sulphate; Analar). The solution was mixed on a heated magnetic stirrer.
Chapter Two 38
1) Tris buffer
0.05 M: Add 12.2 g tris-(hydroxy methyl)-aminomethan AR (MW 121.14) and 74 ml 1 N hydroxychloric acid and dilute to 2 1 with DDW, adjust the pH to 7.6.
0.2 M: Add 2.42 g tris-(hydroxy methyl)-aminomethan AR (MW 121.14) to 100 ml DDW and adjust the pH to 7.4 or 7.6 as required.
m) Acetate buffer
Combine 70 ml 0.2 M sodium acetate (.3H9O; MW 136.08) and 30 ml 0.2 M acetic acid, add 300ml DDW and adjust the pH to 5.0.
n) Tris-maleate buffer
Combine 250 ml 0.2 M tris-maleic acid (6.05 g tris-(hydroxy methyl)- aminomethan AR (MW 121.14) plus 5.8 g maleic acid (MW 116.03) in 250 ml fresh DDW) and 255 ml of 0.2 M sodium hydroxide, adjust pH to 7.2 and store at 4 °C.
o) Glvcerol-gelatin mounting medium
Forty grams of gelatin was dissolved in 210 ml DDW and allowed to stand for 1-2 hours, 250 ml glycerine and 1 g phenol was added, gently heated for 10 minutes while stirring. The mixture was kept at 4 °C and melted prior to use. p) Corpus luteum dissociating medium
This medium was based on calcium- and magnesium-free Hank’s BSS (Flow Laboratories, North Ryde, N.S.W.) with 20 mM Hepes (N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid; Sigma Chemical Co.), 100 U /m l benzyl penicillin G (Commonwealth Serum Laboratories, Melbourne, Vic), 100 Mg/ml streptomycin sulphate BP (Glaxo Australia Pty Ltd, Boronia, Vic), 400 U /m l collagenase (type IV; Sigma) and 1.0 % bovine serum albumin (Sigma). The pH was adjusted to 7.35.
Cnapter Two 39 2.7 CHARACTERIZATION OF CELLS
a) Preparation of single cell suspensions
i) Cells In peripheral lymph
Peripheral lymph was collected as described in section 2.2.d. The lymph was centrifuged at 300 xg for 10 minutes and the plasma aspirated. The plasma was dispensed into polystyrene test tubes (5DT; Disposable Products, Technology Park, SA) and stored at -20 °C. If the RBC content was excessive the cell pellet was resuspended in l-2ml of buffered ammonium chloride (pH 7.3) for 5 minutes. The cell suspension was diluted to three times its volume with PBS prior to centrifugation at 300 xg for 10 minutes. The cell suspension was then washed 2-3 times to remove the erythrocyte debris and finally resuspended in the appropriate medium.
ii) Collection of alveolar macrophages
Following euthanasia the trachea was exposed and it’s cranial end clamped. The trachea and lungs were dissected free and the airways flushed twice with 0.2 % EDTA in PBS. The solution was passed through sterile 80 jum nylon mesh (Henry Simon, North Ryde, N.S.W.) and then centrifuged at 300 xg
for 5 minutes. If necessary, red blood cells (RBCs) were lysed with buffered ammonium chloride solution and washed thoroughly. Cells were resuspended in Eagle’s MEM and adjusted to 1x10^ cells/ml.
iii) Preparation of single luteal cell suspensions
Ovaries were obtained from ewes - under general anaesthesia or immediately following euthanasia and were placed in PBS. Corpora lutea were decapsulated and 0.5 mm sections cut with a scalpel blade. The tissue was placed into round-bottomed 15 ml tissue culture tubes (Disposable Products, S.A.) of known weight. Approximately 1 g of tissue was added to each tube with 5 ml dissociation medium and a 7 mm x 2 mm magnetic stirring bar. The contents of the tubes were stirred for 30 minutes at 32-37 °C. The medium was then decanted, fresh dissociation medium added and the dissociation continued. When tissue slices had decreased to small clumps, the dissociation was hastened by repeatedly drawing the clumps into a pasteur pipette. The dissociation was halted when free cells predominated, usually approximately 2 hours after the process was initiated. The suspension was then passed through 80 ßm nylon mesh (Henry Simon, North Ryde, N.S.W.) and centrifuged at 100 xg for 5 minutes. The supernatant was discarded, and the cells washed 3 times in PBS. The cells
Chapter Two 40 were resuspended in PBS prior to fixation for electron microscopical studies or Hanks BSS with 5 % FCS prior to the production of cytocentrifuged smears.
iv) Preparation? of single lymph node cell suspensions
Lymph nodes were obtained using aseptic technique and placed into sterile PBS. The remainder of the procedure was carried out in a laminar flow hood. The nodes were stripped of fat, their capsules removed and then cut into small sections with scissors. Sections were passed through a wire strainer with the aid of a pestle and rinsed with PBS. Passage through the strainer was repeated until coarse node fragments were removed. The cell suspension was then passed through an 80 ßm nylon mesh (Henry Simon, North Ryde, N.S.W.). The suspension was centrifuged at 300 xg for 5 minutes and resuspended in PBS twice prior to resuspension in culture medium.
v) Peripheral blood lymphocyte (PBL) separation using density gradient
media
Jugular venous blood was collected into tubes containing EDTA or heparin. The whole blood was centrifuged at 840 xg for 25 minutes and the buffy coat aspirated. The buffy coat was resuspended in twice its volume of PBS, layered over Ficoll-Hypaque (Ficoll-Paque, Pharmacia Fine Chemicals, Uppsala, Sweden; Density 1.077 g/ml) and centrifuged for a further 25 minutes at 840 xg. The lymphocyte rich layer above the Ficoll-Hypaque was then collected, resuspended in PBS, centrifuged at 300 xg for 10 minutes and the supernatant aspirated. The cell pellet was resuspended in 2-3ml of buffered ammonium chloride (pH 7.3) for 2-5 minutes to lyse the erythrocytes. The cell suspension was diluted to three times the volume with PBS prior to centrifugation at 300 xg
for 10 minutes. The cell suspension was then washed with PBS 3-4 times to remove the erythrocyte debris and finally resuspended in the appropriate medium.
vi) PBL enrichment by erythrocyte lysis
Jugular venous blood was collected into tubes containing EDTA or heparin. The whole blood was centrifuged at 840 xg for 25 minutes and the buffy coat aspirated. The buffy coat was resuspended in twice its volume of PBS, centrifuged at 300 xg for 10 minutes and the supernatant aspirated. The cell pellet was resuspended in 2-3ml of buffered ammonium chloride (pH 7.3) for 2-5 minutes to lyse the erythrocytes. The cell suspension was diluted to three times the volume with PBS prior to centrifugation at 300 xg for 10 minutes. The cell
Chapter Two 41
suspension was then washed with PBS 3-4 times to remove the erythrocyte debris and finally resuspended in the appropriate medium.
vii) B lymphocyte enrichment of PBL
Antibody solution (10 ml) was placed into plastic petri dishes (90 mm diameter, Bunzl Medical and Laboratory Products, Seaton, S.A.) which were then incubated overnight at 4 °C. The antibody solution used contained F(abT)2
fragments of rabbit anti-sheep IgG (heavy and light chains specific; Cappel Scientific Division, Cooper Biomedical, Inc., Malvern, USA) diluted to 5 /xg/ml in PBS.
Dishes were washed gently five times with 10 ml PBS just prior to the addition of the lymphocyte suspension. The PBL suspension was adjusted to 20 xlO^ cells/ml in PBS with 5 % FCS and 80 xlO^ cells (4 ml) were added to each dish. Dishes were incubated at room temperature for 1 hour with agitation for 30 seconds after 30 minutes.
After incubation, the B cell depleted fraction was decanted and collected, and the dishes were washed four times by gently pouring PBS with 1 % FCS onto each dish to remove residual non-adherent cells. Adherent cells were removed by flooding the dishes with 20 ml of PBS with 1 % FCS and systematically flushing the surface of each dish, in strips 1-2 cm wide, using a pasteur pipette. The medium was collected and the flushing process repeated. Smears of adherent and non-adherent cells were made with a cytocentrifuge.
b) Cell counts and cell outputs
An electronic cell counter (Coulter Model FN, Coulter Electronics Ltd., Dunstable, Beds., England) was used to determine the total number of RBCs, white blood cells (WBCs) and large WBCs in lymph. A haemocytometer (Double Neubauer; Clay Adams, Parsippany, USA) was used to count the total number of WBCs in peripheral blood following a 1:10 dilution in WBC counting fluid.
Volumes of lymph were estimated by weight and the lymph flow rate per hour for each collection period was recorded. The average cell output per hour was calculated from this volume and the cell content. Allowance was made for the anticoagulant solution infused into the lymphatic cannulas in these calculations.
Chapter Two 42 c) Cell smears
White blood cell differential counts were performed on Diff Quik stained whole blood smears. Lymph cells were separated from lymph plasma by centrifugation at 300 xg for 10 minutes, washed twice in PBS and resuspended in Hanks BSS at a concentration of 1 xlO^ cells/ml. If excessive numbers of RBCs were present the cell pellet was resuspended in buffered ammonium chloride solution prior to washing with PBS. Cytocentrifuge smears (Cytospin; Shandon Elliot) were obtained from 100 ß\ of the cell suspension. The smears were stained using Diff Quik after air drying.
d) Cell viability
The trypan blue exclusion test was used to assess cell viability. A stock solution of 4 % trypan blue was further diluted with PBS to 0.4 %. One drop of dye was added to 3 drops of cell suspension and allowed to stand for at least 10 minutes at 37 °C. The proportion of dead cells was determined by counting 200 cells in a haemocytometer.
e) Histology
i) Paraffin sections
Tissue samples, 1 cm or less in thickness, were fixed in 10 % neutral buffered formalin for a minimum of 2 days. The fixed tissue was automatically processed with an Autotechnicon DUO model 2A (Technicon Ltd, Ireland) and embedded in Paramat (Gurr BDH Chemicals Ltd, Poole, England). Sections 4 /im in thickness were taken from the embedded tissue with a Reichert 2050 microtome (Cambridge Instruments GmbH, West Germany) and then mounted and stained with giemsa or toluidine blue.
ii) Frozen Sections
Tissue sections were cut to 2 mm in thickness and quick-frozen in hexane at -60 °C for one minute. Cryostat (Bright 5030 microtome, Bright Instrument Co. Ltd., Huntingdon, England) sections 8 ßm in thickness were cut and placed on gelatin-coated glass microscope slides. They were stored overnight in a dessication chamber at 4 °C prior to immunohistochemical or histochemical procedures.
Chapter Two 43
f) Transmission electron microscopy
A cell pellet was obtained by centrifugation at 300 xg for 10 minutes and if necessary RBC’s were lysed with buffered ammonium chloride solution followed by two washes in PBS. The washed cells were fixed in 3 ml of 3 % glutaraldehyde in 0.1 M cacodylate for 3 hours. The cells were centrifuged and
tetroxide resuspended in 0.1 M cacodylate buffer. They were post-fixed in 1 % osmium tor 1.5 hours and, after 3 washes in distilled water, suspended in 1 % uranyl acetate for 1.5 hours. Following a further 3 washes in distilled water, the specimen was dehydrated in graded alcohol solutions, embedded in Spurrs resin (Polaron Equipment Ltd., Hertfordshire, England), sectioned on a Reichert Ultracut E (Cambridge Instruments GmbH, West Germany) and stained with Reynold’s lead citrate and uranyl acetate. The stained sections were examined in a Philips 301 Electron Microscope (Holland).
g) Histochemical techniques
i) Acid phosphatase
This enzyme was demonstrated by Gomori’s lead nitrate method (Pearse 1968) with slight modification (Landsverk 1980). Smears were fixed in 10 % formol-calcium for 5 minutes at 4 °C and washed three times in DDW. This was followed by a 30 minute incubation at 37 °C in the substrate solution which was prepared thus: 306 mg of sodium-/J-glycerophosphate was dissolved in 100 ml acetate buffer at 37 °C; when completely dissolved 100 ml 4 mM lead nitrate was added; some precipitate occurs; the solution was allowed to stand for 15-30 minutes and then filtered immediately prior to use. After the incubation, the smears were washed 3 times in DDW and then placed in 1 % ammonium sulphide in DDW for 1-2 minutes. Three washes in DDW preceded staining for contrast and mounting glycerol-gelatin.
ii) Magnesium adenosine triphosphatase (Mg^ + -ATPase)
Mg ATPase was demonstrated by a modification of the Wachstein and Meisel (1957) technique (Jacobsen and Jorgensen 1969). Specimens were fixed in 10 % formol-calcium at 4 °C for 5 minutes. The slides were washed in 3 changes of 0.2 M Tris maleate buffer, pH 7.2. The incubation medium was prepared as follows: 90 mg of adenosine triphosphate (ATP) was dissolved in 20 ml DDW, adjusted to pH 7.2 and then 20 ml 0.2 M Tris maleate buffer, 3 ml 2 % lead nitrate, 3.7 ml 1 % magnesium sulphate and 3.3 ml DDW were added. Tissue sections were incubated for 20 minutes and smears for 40 minutes. The
Chapter Two 44 specimens were washed three times in DDW and then placed in 1 % yellow ammonium sulphide (BDH Chemicals Ltd.) for 30 seconds to 2 minutes. The specimens were counterstained with methyl green and mounted with glycerol- gelatin. The four controls were as follows: heat inactivation of enzyme at 80 °C in DDW for 10 minutes; omission of magnesium from the medium; complete medium with 100 mM sodium fluoride as an inhibitor; and replacement of ATP with equimolar concentrations of adenosine diphosphate or ^-glycerophosphate. With the magnesium deficient medium demineralised water was always used.
iii) Non-specific esterase
The method of Koski, Poplack and Blaese (1976) was followed except for the substitution of a-naphthyl acetate for a-naphthyl butyrate (Yang et al 1979). The slides were placed in fixative for 30 seconds at 4 °C and washed three times in DDW. They were then incubated with a mixture of hexazotized pararosaniline and a-naphthyl acetate for 45 minutes at 37 °C. The substrate solution was prepared using the following solutions:
Hexazotized pararosaniline - combine equal portions of freshly filtered pararosaniline solution (1 g pararosaniline hydrochloride [Sigma Chemical Co] and 25 ml 2 M HC1 stored at 4 °C) and 4 % sodium nitrite (100 mg sodium nitrite in 2.5 ml DDW prepared fresh for each use, and allowed to stand for one minute before use).
M/15 Sorenson’s phosphate buffer (pH 7.6) - 2.128 g Na2HP0 4, 6.984 g KH2P 0 4 and 11 DDW.
a-naphthyl acetate solution - 1 g a-naphthyl acetate (Sigma Chemical Co) and 50 ml ethylene glycol monomethyl ether mixed in a glass bottle using glass pipettes; stored at -20 °C for up to 2 weeks and protected from light.
The mixture .was obtained by adding, in sequence, 44.5 ml M/15 Sorenson’s buffer, 0.25 ml hexazotized pararosaniline and 3 ml a-naphthyl acetate solution. This solution was passed through a 10 /xm millipore filter prior to use.
The slides were then washed 4 times in DDW and counterstained with methyl green. Specimens were dehydrated by passage through ethanol to xylene and mounted with Histoclad (Clay Adams, New Jersey, U.S.A.).
Chapter Two 45
h) Immunohistochemial techniques
i) Alkaline phosphatase anti-alkaline phosphatase
All samples were fixed in 10 % formol-calcium at 4 °C for 5 minutes, followed by three washes in 0.05 M Tris-buffered saline (TBS), pH 7.6. All subsequent procedures were performed at room temperature (about 22 °C). A 1 in 2 dilution of non-immune rabbit serum in 0.05 M TBS, pH 7.6, was applied for 20 minutes and then drained prior to application of the mAb. The specimens were incubated for 30 minutes with the appropriate dilution of mAb ST-1 (Beya
et al 1986) and SBU-T1 (Mackay et al 1985), or alkaline phosphatase labelled
rabbit anti-sheep IgG (heavy and light chain; Kirkgaard and Perry Laboratories Inc., Gaithersburg, U.S.A.), in 0.5 % swine gelatin in 0.05 M TBS, pH 7.6. After washing three times in 0.05 M TBS, a second antibody, rabbit anti-mouse immunoglobulin (Dakopatt Z259), diluted 1 in 25, was applied for 30 minutes to those slides labelled with mAb. After 3 washes in TBS mAb labelled slides were incubated for 30 minutes with a 1 in 20 dilution of soluble complexes of alkaline phosphatase and mouse mAb anti-alkaline phosphatase (Dakopatt D651). The samples were washed again and then incubated for 30 minutes with the filtered substrate solution which was composed of: 50 ml 0.2 M TBS, pH 7.6; 15 mg naphthol AS-MX phosphate (Sigma Chemical Co.) (dissolved in 0.5 ml N,N dimethyl formamide (Mallinckrodt, Australia)); 40 mg fast red violet LB salt (Sigma Chemical Co.); and 12 mg levamisole (Sigma Chemical Co.). After a further washing the frozen sections and cytocentrifuge smears, not subject to enzyme histochemical procedures, were counterstained with methyl green and mounted with glycerol-gelatin.
i) Flow microfluorometry
i) Labelling of cells
Reagent antisera and mAbs were diluted to appropriate concentrations in PBS with 0.1% sodium azide immediately before use, and centrifuged in a Beckman microfuge (approximately 9,000 xg\ Model B, Beckman Instruments Inc., Palo Alto, CA. USA) for 5 minutes.
Approximately 1 xlO^ cells were incubated with 100 p\ of optimally diluted mAb at 4 °C for 20 minutes. The cells were then incubated with 100 /d of a 1 in 50 dilution of affinity-purified fluorescein-conjugated sheep anti-mouse IgG antibody at 4 °C for 20 minutes. After each incubation the cell suspension was underlaid with FCS, centrifuged at 300 xg for 5 minutes at 4 °C (Model TJ-6 centrifuge with a TJ-R refrigeration unit, Beckman Instruments Inc., Palo Alto,
Chapter Two 46
CA. USA) and the supernatant aspirated. The cells were given a final wrash in PBS and 0.1% sodium azide, centrifuged at 4 °C and the supernatant aspirated. The cells were resuspended in either Eagles MEM with 3% FCS and 0.1% sodium azide and analysed on the same day or 1% paraformaldehyde in normal saline and stored at 4 8C overnight prior to analysis.
The labelled cells were analysed on a Fluorescence Activated Cell Sorter IV (FACS; Becton Dickinson, Mountain View, CA. USA). Viable lymphocytes were distinguished from monocytes, granulocytes, dead cells and debris on the basis of their forward versus 90° light scatter. An electronic two-dimensional