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CHAPTER TWO MATERIALS AND METHODS
Molecular biology
DNA techniques
To propagate DNA for transfection or cloning, DNA was transformed into XL10-Gold ultracompetent bacterial cells (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Transformed bacteria were spread onto 20 g/L LB Broth / 20 g/L Agar (both GE Healthcare, Buckinghamshire, UK) with appropriate selection antibiotics (50 μg/mL ampicillin; 30 μg/mL kanamycin, both Sigma-Aldrich, St Louis, MO). Isolated single colonies were picked from transformation plates and incubated in LB Broth with appropriate selection antibiotics for 16 h at 37°C with shaking to promote aeration. DNA was isolated and purified with Mini-Prep (Qiagen, Hilden, Germany), or Purelink HiPure Midi-Prep (Invitrogen, Carlsbad, CA) kits according to the manufacturer’s instructions. Bacterial cultures were 8 mL for Mini-Prep or 50 mL inoculated with 50 μL from an 8 mL starter culture for Midi-Prep. As required, DNA was visualised by agarose gel electrophoresis.
DNA concentrations were quantified with a NanoDrop™ 1000 spectrophotometer (Thermo Scientific, Waltham, MA). For experiments in which DNA concentration was imperative three measurements were taken and averaged. DNA constructs were sequence-verified (DNA Sequencing and Genotyping Facility, School of Biological Sciences, University of Auckland, NZ) using “universal” primers that aligned with sequences in the flanking regions of the plasmid multiple cloning site. Sequencher™ (v. 4.9, Gene Codes, Ann Arbor, MI) and ChromasPro (v. 1.32, Technelysium, Australia) software was used to assess sequencing results and plan cloning strategies.
The HA-hCB1 and HA-hD1 plasmids were purchased in vector pcDNA3.1(+) (Invitrogen). Donor and acceptor (pEF4/V5-His A, Invitrogen) plasmids were digested with KpnI/PmeI (CB1) or KpnI/XbaI (D1) restriction enzymes (KpnI and XbaI: Roche, Mannheim, Germany; PmeI: New England Biolabs, Ipswitch, MA), agarose gel purified (QIAquick gel extraction kit, Qiagen) and ligated with T4 DNA ligase (Invitrogen) according to the manufacturers instructions. Use of the KpnI/PmeI restriction enzyme pair resulted in excision of the V5 and poly-His epitopes from the pEF4a vector backbone.
The pplss HA-hCB1 construct was generated by a colleague (Dr Emma Scotter) for use in a parallel project. Briefly, primers designed with Primer3 (v. 0.4.0, http://frodo.wi.mit.edu/, Rozen and Skaletsky, 2000) were utilised to PCR amplify a 56 base pair pplss coding region from a plasmid gifted to the lab by Prof. Ken Mackie. The PCR reaction included KpnI restriction sites in the flanking regions of the pplss sequence which were subsequently used to insert the pplss into the HA-hCB1 in pEF4a plasmid, N-terminal and in-frame with the HA epitope sequence.
EGFP-Rab GTPase constructs were also utilised. These are described in detail in chapter five (pg. 118)
Cell cultur
Cell lines
The cell lines utilised and DNA constructs they were transfected with (as applicable) are listed in Table 2.2. 1(Shaw et al., 2002); 2ATCC, American Type Culture Collection; 3(Tooze et al., 1989); 4(Jordan et al., 2005; Graham et al., 2006).
Routine maintenance
Cells were maintained at 37°C / 5% CO2 in Dulbecco’s modified eagle’s medium (DMEM; Invitrogen) with 10% FBS (NZ-origin, Invitrogen), except the CHO cells which were maintained in DMEM-F12 (Invitrogen) with 10% FBS. Transfected lines were cultured with 250 μg/mL Zeocin™ (Invitrogen) to promote continued heterologous receptor expression. The Neuro-2a cell media was buffered with 25 mM HEPES pH 7.4.
Cells were cultured in 25cm2 or 75cm2 filter capped, canted neck flasks (BD Biosciences, San Jose, CA). Upon reaching 80-100% confluency (approximately every 2-3 days), cells were sub-cultured with a split of not more than 1:15 for CHO cells or 1:6 for the other cell types. For sub-culturing, cells were rinsed with phosphate buffered saline (PBS; NaCl 1.4 M, KCl 27 mM, Na2HPO4 81 mM, KH2PO4 15 mM), incubated at 37°C with 0.05% trypsin-EDTA (Invitrogen) for 3-5 min, triturated in media and transferred into a new flask with fresh supplemented media.
Cryogenically frozen cell stocks were stored for the long-term preservation of cells at low passage numbers. Fresh cells were thawed periodically or in the event a change in cell behaviour or morphology was noted to avoid experiments being influenced by potential changes in cell phenotype over time.
To create frozen stocks, cells were trypsinised, re-suspended in supplemented media, pelleted at 150 xg for 5 min and re-suspended in ice-cold FBS containing 10% dimethylsulfoxide (DMSO; J.T.Baker). Re-suspended cells at a concentration of 5-10 million cells/mL were transferred to cryovials (Greiner Bio-One, Kremsmuenster, Austria) and stored at -80°C for 1-3 days prior to long-term storage in liquid nitrogen.
Frozen stocks were revived by thawing rapidly to 37°C in a waterbath, re-suspending in supplemented media, pelleting the cells (to remove residual DMSO), re-suspending again in media, and transferring to a culture flask for maintenance. Media was changed and selective antibiotics added approximately 24 h later.
Generation of stable cell lines
All the transfected cell lines listed in Table 2.2 were transformed such that the introduced receptor was expressed stably over time. DNA was linearised with ScaI restriction enzyme (Roche) to facilitate genomic DNA incorporation and transfected with Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s recommendations. Briefly, 2 µL Lipofectamine™ 2000 per 100 uL Opti-MEM® (Invitrogen) was mixed and incubated at room temperature (RT) for 5 min. 8 ng/µL DNA was mixed with Opti-MEM®. The Lipofectamine™ 2000 and DNA mixes were then combined in equal volumes and incubated at RT for 20 min prior to 100 μL being added dropwise to cells that had been seeded (see below) in a 24-well plate and reached 90-100% confluency. 24 h post-transfection, cells were transferred to a 6-well plate. Another 24 h later selection antibiotics were added (350 μg/mL Zeocin™). In each transfection a no-DNA control was included so that death of cells not expressing the transfected construct could be monitored.
Following the death of the control cells (after approximately 2 weeks of maintenance in selection media), transfected cells were plated sparsely in a 6-well plate and allowed to grow in clonal colonies. Cells were labelled with primary and secondary antibodies to detect surface receptor (see below), and observed under a fluorescent microscope. Colonies expressing detectable levels of receptor were gently transferred into new culture dishes. As picking could not be performed under sterile conditions, cells were cultured with Penicillin-Streptomycin (Invitrogen) for 1 week to prophylactically prevent bacterial contamination. Surface and total receptor expression, and the clonal nature of the resultant cell lines were subsequently confirmed with standard immunocytochemistry.
Cell plating for experiments
Cells were seeded at an appropriate density to reach 70-80% confluence by the end of the experiment and allowed to recover overnight. Following trypsinisation (as above), a small aliquot of cells diluted in trypan blue (0.4%; Invitrogen) was counted in a haemocytometer. Cells were diluted appropriately in supplemented media and dispensed into a culture vessel with regular agitation to promote even distribution of cells between wells. For HEK cells a plating density of 26,000 to 30,000 cells per well in a 96-well plate would produce a confluency of 70-80% 24 h after seeding.
To aid cell adherence for trafficking or immunocytochemistry experiments, culture vessels were pre-treated with Poly-L-Lysine (0.2 mg/mL in PBS, Sigma-Aldrich) prior to cell seeding. Vessels were instead treated with Poly-D-Lysine (0.05 mg/mL in PBS, Sigma-Aldrich) if multi-chamber glass culture slides were used (for confocal imaging; BD Biosciences) or for experiments in which cells were to be treated with trypsin. Poly-L or Poly-D-lysine was incubated on the plastic or glass at 37°C for at least one hour, following which the plate was rinsed once with PBS
Trafficking assays
Due to their lipophilicity, cannabinoids exhibit a tendency to be adsorbed by similarly hydrophobic surfaces, such as plastic, and have affinity for a number of the components in full-serum. Therefore, to promote the maintenance of cannabinoid drugs in solution, vessels used to dilute or dispense drugs were silanised prior to use (Coatasil, Ajax Finechem, Sydney, NSW) and drugs were diluted in serum-free media (SFM; DMEM or DMEM-F12 with 5 mg/mL BSA, ICPbio, Auckland, NZ; see also Hillard et al., 1995).
Prior to the start of each assay, cells were equilibrated in SFM for 15 min. The details of drug stimulations are noted in the text and were performed at 37°C unless otherwise stated. Table 2.3 lists the drugs and chemicals utilised. At the conclusion of drug stimulation, plates were placed on ice to prevent any further receptor trafficking, processed for immunocytochemistry as appropriate, and fixed (4% paraformaldehyde in 0.1 M phosphate buffer [PFA], 10 min at RT, followed by three PBS washes).
Vehicle and washing controls were included with each experiment. Final ethanol concentrations did not exceed 0.1%. A minimum of three, but regularly 4-5 replicate wells were included in each quantified experiment. The positioning of timepoints or drug conditions in 96-well plates was randomised for each experiment. In order to avoid effects of uneven evaporation from the edges of culture plates, the outside wells were filled with “sacrificial” media and not assayed in the experiment. Except when rapid cooling was intended (at the end of an experiment and/or for immunocytochemistry protocols), cells were kept at a constant temperature by incubating in a 37°C incubator during long stimulations. When cells were removed from the incubator to add drugs, plates were placed on a polystyrene surface to prevent conduction of heat from the bottom of the plate. Experiments requiring frequent drug treatments over period of 30 min or less (internalisation) were performed with the cell culture plate sitting on the surface of 37°C waterbath to maintain a constant temperature.
If intracellular receptors were to be assayed in the experiment, prior to fixation or immunocytochemistry, cells were incubated with 0.05% trypsin-EDTA for 1 min at RT. Control cells for comparison were incubated with 0.2 g/L EDTA•4Na alone. Activity of the trypsin enzyme was halted by adding an equal volume of full-serum media or 2 mg/mL trypsin inhibitor from soybean (TIS; Sigma-Aldrich). Optimisation of this method is described in chapter three (pg. 64)
1 Introduction
G-Protein Coupled Receptor Intracellular Trafficking
Intracellular trafficking pathways
Control and modulation of receptor trafficking
GPCR trafficking in disease
Cannabinoid Receptor 1 Function, Pharmacology and Intracellular Trafficking
In vivo functions and implications in disease
Signalling
Aims and Hypotheses
2 Materials and Methods
Molecular biology
Cell culture
Trafficking assays
Immunocytochemistry
Imaging and quantification
cAMP assays
Western blotting
Data presentation and statistic
3 Quantitative Assay Development
Introduction
Methods
Colocalisation
Image acquisition with Discovery-1™
Assessment of receptor internalisation by Granularity.
Assessment of receptor expression by Total Grey Value per Cell
4 Investigations into CB1 Trafficking and the Role of the Intracellular Pool
Introduction
Methods
Results
Discussion
5 Rab GTPase Modulation of CB1 Trafficking
Introduction
Methods
Results
Discussion
6 Discussion, Conclusions and Future Directions
Method optimisation
7 Appendices
8 References
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