2.2.1 Cell Culture
Rbl-2H3 cells (kind gift from Mark Marsh, MRC Unit, Laboratory for Molecular Cell Biology) were grown as an adherent monolayer in Dulbeco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 50|Lig/ml gentamicin, at 37°C, 5% CO2.
HUVEC (Cascade Biologies Inc.) were grown on gelatin-coated dishes in
Medium199 (M199), 20% fetal calf serum (characterised, triple 0.1 p,m filtered, HyClone), lOU/ml heparin, 3mg/ml endothelial cell growth supplement (ECGS) and 50|ig/ml gentamicin, at 37°C, 5% CO2.
For both cell types, cells were passaged by rinsing in warm (37°C) PBS, then trypsinised and plated out in full growth medium.
2.2.2 Transient Transfection
Electroporation of Rbl-2H3 cells Approximately 4x10^ cells were electroporated
(three pulses) at 125microfarads, 250Volts, infinity ohms. Either 3^ig or lO^ig plasmid DNA was used for transfection. Cells were replated in full growth medium and typically analysed three days post-transfection.
Nucleofection of HUVEC The amaxa biosystems HUVEC Nucleofector™ Kit was
used for transfection of HUVEC according to the manufacturers instructions. Briefly, cells on 15cm dishes were rinsed with PBS, trypsinised and pelleted. 1-5 xIO® cells were used per Nucleofection and resuspended in lOO^il Nucleofection buffer (at room temperature) with 5|Lig plasmid DNA. This solution was pulsed in the Nucleofector™, using Program U-01 and cells were immediately transferred to pre-warmed (37°C) full medium and plated onto gelatin-coated dishes.
2.2.3 Immunofluorescence Microscopy
All manipulations were carried out at room temperature. Cells on 10mm glass coverslips were fixed in 3% paraformaldehyde (in PBS) for 15 minutes and then quenched and permeablised in 50mM NH4CI and 0.2% saponin (in PBS) for 20 minutes. Coverslips were then rinsed twice with PBS and transferred to PGS (PBS, 0.2% gelatin and 0.02% saponin). Primary antibody was incubated in PGS for 1 hour followed by 3 washes in PGS. Secondary fluorophore-conjugated antibody was then incubated in PGS for 30 minutes. Labelled cells were then rinsed three times in PGS, three times in PBS, and then dunked in water before drying and mounting in 6.5pl Mowial.
For cells that possessed HRP to be monitored by fluorescence microscopy, the quenching and permeabilisation steps were separated such that cells were permeablised following Tyramide Signal Amplification (TSA) cyanine 3 (Perkin Elmer Life Sciences) treatment. Subsequent to quenching of the fixative, coverslips were rinsed in PBS and placed in TSA reagent for 2-10 minutes. HRP activity was stopped by transferring coverslips to PBS plus 0.2% azide and rinsed with this solution 10 times. Permeabilisation then ensued, followed by antibody incubations in PGS.
Mowial was prepared by mixing 2.4g Mowial and 6g glycerol. 6ml H2O was added and the solution left for several hours at room temperature. 12ml of 0.2M TrisHCI pH8.5 (final concentration ~7mM) was added to the solution and heated to 50°C for a few hours. Sonication for a few hours was necessary to dissolve all the Mowial. Once dissolved, Mowial was clarified by centrifugation at 5 OOOg for 15 minutes, aliquoted out and stored at -20°C.
Mounted coverslips were analysed by confocal microscopy with the use of an Optiphot-2 microscope (Nikon, Tokyo, Japan) equipped with a MRC Bio-Rad 1024 confocal laser scanning system. Images were transferred to Adobe Photoshop (Adobe Systems, Mountain View, CA).
2.2.4 Antibodies
Antibodies routinely used for fluorescence microscopy were rabbit-anti-serotonin (Biogenesis), mouse-anti-CD63 / 1B5 (kind gift from Mark Marsh, Fraile-Ramos et al. 2001), mouse-anti-vWF (Serotec), rabbit-anti-vWF (Dako), sheep-anti-vWF
(Serotec), rabbit-anti-P-selectin (Fonzie, Cutler lab), rabbit-anti-cathepsin D (kind gift from Collin Hopkins, Imperial College), rabbit-anti-lgp120 (kind gift from Collin Hopkins, Imperial College), mouse-anti-GFP (Sigma), sheep-anti-GFP
(Biogenesis). Secondary donkey antibodies against sheep, rabbit and mouse conjugated to FITC, or Texas Red were from Jackson Immunoresearch.
2.2.5 r^HI-Serotonin Loading
Rbl-2H3 cells were loaded by overnight incubation of 0.2pCi/ml [^H]-serotonin (hydroxytryptamine binoxylate-5) in full growth medium. Cells were then rinsed twice and further incubated for 1 hour in full medium. [^H]-serotonin incorporation was monitored by tritium radioactivity using liquid scintillation spectrometry.
To inhibit the vesicular uptake of [^H]-serotonin, 10|Lig/ml reserpine was incubated overnight with cells in addition to 2.0p,Ci/ml [^H]-serotonin.
2.2.6 r^^^ll-Transferrin Loading
Rbl-2H3 cells were serum starved (DMEM, 10mM HEPES, 2mg/ml BSA) for 1 hour and then incubated with O.G^Ci/ml [^^®l]-transferrin in serum-free medium for a further 1 hour, either at 37°C or at 4°C. To compete the specific uptake of
0.6pCi/ml [^^^l]-transferrin, 0.12pg/ml unlabelled holotransferrin was also added. The radio-label was then washed from the cells by rinsing three times with ice cold DMEM.
To locate the plasma membrane, cell surface [^^^l]-transferrin was stripped by the following method. Subsequent to loading, cells were transferred to ice, rinsed, and incubated for 15 minutes in 20mM sodium acetate, pH5, 2mM CaCb, 150mM NaCI, and 50|xM deferoxamine mesylate. This solution was then replaced with PBS+ (PBS, ImM MgCb, 0.1 mM CaCl2) and further rinsed and incubated for 20 minutes with PBS+ and 50|iM deferoxamine mesylate.
[’'^^Ij-transferrin loading was monitored by gamma counting using a Packard Cobra II auto-gamma counter.
2.2.7 HRP Loading
High grade HRP (Type II) was used to load cells at a concentration of 5mg/ml in full medium for 15 minutes. Cells were washed twice with full medium and then incubated for at least 4 hours to chase the HRP into the lysosomes. HRP loading was monitored using the HRP-OPD and HRP-fluorescence assays described
below.
2.2.8 Stimulation of cells
Rbl-2H3 cells were primed overnight with O.S^ig/ml anti-dinitrophenol IgE
(monoclonal, SPE-7) in full Rbl-2H3 growth medium prior to stimulation studies the following day. Cells were then rinsed twice and cultured for a further 1 hour.
Stimulation was induced by the addition of 50ng/ml human serum albumin/2,4- dinitrophenol to cross-link the IgE bound to the FceRI on the cell surface for 25 minutes at 37°C in DMEM (lacking phenol red, without serum, lOmM HEPES, 2mg/ml BSA). Cells were then placed on ice, the bathing medium removed and kept for analysis, and the cells prepared for subcellular fractionation, see below.
2.2.9 Subcellular Fractionation
Sucrose equilibrium gradients Rbl-2H3 cells to be fractionated were placed on
ice and rinsed twice with homogenisation buffer, HB (250mM sucrose, ImM MgCl2, lOmM HEPES pH7.3). Cells (4x10^) were then scraped in 1.5ml HB (plus ImM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail). The cell
suspension was homogenised by five passages through a ball bearing
homogeniser with 0.009mm clearance (EMBL, Heidelberg, Germany). The nuclear fraction was spun down at 170g for 10 minutes at 4°C and then 800units/ml
DNasel (EC3.1.21.1) was added to the post-nuclear supernatant (PNS). The PNS was loaded onto a pre-formed continuous sucrose gradient of 0.45-2.0M sucrose (10ml) with a 300p.l 70% sucrose cushion, and run at 29,900rpm for 18 hours at 4°C. Gradients were collected in 25 fractions of 500pl from the top of the tube using an Autodensi-Flow IIC (Buchler Instruments, Kansas City, MO).
For experiments that required a further secondary gradient, fractions 14-20 were analysed and the remainder pooled to 4ml at 1.1 M sucrose. This was then layered
on a pre-formed continuous gradient of 1.3-2.0M sucrose (8ml,) and run as with the first gradient.
Percoll equilibrium gradients HUVECs were PBS rinsed and pelleted by
trypsinisation. Cell pellets were resuspended in 800|il HB plus 800U/ml DNasel (EC3.1.21.1) on ice, and homogenised by ten passages through the ball bearing homogeniser. The PNS was obtained by spinning at 170g at 4°C for 7.5 minutes and mixed with 3ml of Percoll stock (250mM sucrose, lOmM HEPES pH7.3 and 89% Percoll). The remainder of the volume in a 5.1ml Beckman tube was made up with HB and the contents were mixed. Gradients were run at 30,000rpm, for 1 hour at 4°C and fractionated into 12 fractions of 425)liI.
When necessary, Percoll was spun out of the fractions by the following method. 300pl of each fraction was transferred into a 1.5ml Beckman tube and 27^1 of 12%TX114 plus protease inhibitors was added to give a final concentration of 1%TX114. Samples were left on ice for 10 minutes to solublilise with occasional vortexing and then spun at 43,00rpm for 2 hours at 4°C. The TX114 insoluble precipitate was transferred to a fresh tube, as was the 1%TX114 supernatant. 500|liI H2O was added to the 1%TX114 supernatant to dilute out any remaining Percoll and the solution was partitioned as described later in TX114 partition.