Material( Supplier(
2.7 Bioassays
2.7.1 Transwell migration assays
Migration assays were performed with MSCs. 10,000 MSCs were seeded on to PET
membrane transwell inserts (BD Biocoat) containing 8µm pores in 500µl assay
media. The inserts were placed into wells containing 750µl of the appropriate media
and incubated for 16h. Following incubation, the cells that remained inside the insert were removed using a wet cotton bud. Cells that had migrated to the underside of the transwell inserts were fixed and stained with the Reastain Quickdiff kit (Reagena) according to the manufacturers instructions. Membranes were then cut from the transwell inserts and mounted on glass slides in immersion oil for imaging using a Leica DMIL microscope with the acquisition software Leica Application Suite. Cells were counted as an average of 10 independent fields at 10X magnification (figure 4.1) or counted in 15 fields at 20X (general pattern shown in figure 2.2) and summated. Cells that over-‐lapped fields on the right side were ignored.
Figure 2.2. 15 fields of migrated cells that were counted at 20X following transwell migration assays
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2.7.2 Cell proliferation assays
2.7.2.1 Detection of EdU incorporation by flow cytometry.
Treatments were exposed to 10μM 5-‐ethynyl-‐2´-‐deoxyuridine (EdU) for the desired time (10 or 16 h). Following exposure, cells were trypsinised, washed in 1% BSA and fixed using 4% paraformaldehyde for 10 min at room temperature. Cells were washed once with 1% BSA and then washed with Click-‐iT saponin-‐based permeabilisation and wash reagent. Afterwards, fixed samples were incubated with 500μl Click-‐iT reaction cocktail (prepared according to manufacturers instructions) and incubated in the dark for 30 min at RT. Cells were washed with Click-‐iT saponin-‐
based permeabilisation and wash reagent and re-‐suspended in 200µl to be analysed
using a FACScalibur (BD Biosciences) flow cytometer on a low flow rate.
2.7.2.2 Detection of EdU incorporation in direct and transwell co-‐
cultures.
AGS-‐ or MKN45-‐dTomato cells were exposed to assay media for 48h prior to seeding co-‐cultures to synchronise AGS/MKN45 cell-‐cycles. To set-‐up co-‐cultures, 2.4x104/cm2 labeled cancer cells with or without 4.8x103/cm2 MSCs, CAMs or cancer
cells (to control for cell numbers) were seeded in triplicate into 6cm dishes with
assay media. Fourteen hours later, 10μM EdU was added to each dish and incubated for 10 hours. Samples were prepared for analysis as described in 2.7.2.1. EdU incorporation was only assessed in cells expressing dTomato.
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2.7.2.3 3D growth assay
In vitro tumour cell growth was monitored using a 3D growth assay based on the methods published by (Sasser et al., 2007). This method involves seeding EGFP-‐ labeled cells into a 3D matrix of phenol red free, growth factor reduced matrigel (BD Biosciences), whereby relative growth can be monitored over an extended period of time. Fluorescently tagged cells were produced using a lentivirus expressing EGFP at a MOI of 10. 1x105 GFP-‐tagged cells with or without 2x104 MSCs/myofibroblasts
were suspended in 400µl phenol red-‐free αMEM 1% GlutaMax 2% FBS and 4mg/ml matrigel and placed (in triplicate) into a well of a 96 well black-‐walled tissue culture plate (Porvair Sciences) avoiding bubble formation. Cultures were incubated at 37oC
5% CO2 for 5 hours before the addition of 50µl phenol red-‐free αMEM 1% GlutaMax
2% FBS. Immediately after, a day 0 fluorescent reading using a GloMax®-‐Multi Detection System (Promega) was taken. Relative fluorescence units (RFU) were normalised to identical unlabeled cell controls and the plate was analysed each day for 6 days. Before reading, plates needed to be cooled at RT for 15 minutes to prevent condensation.
2.7.3 Collagen contraction assay
Cells were trypsinised and re-‐suspended in DMEM 8% FBS at a density of 80,000
cells/ml. A collagen matrix solution was prepared on ice consisting of 800µl collagen
I (3.75mg/ml), 100µl 10X DMEM, 80µl H2O and 20µl 1M NaOH per sample. 1ml cell
suspension was added to the collagen solution, mixed thoroughly (with care taken to
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matrices were incubated for 2 hours to allow polymerisation before being detached
using a 200µl tip and 500µl serum-‐free DMEM was added to each well to reach a
final FBS concentration of 2%. 24hs later, wells and collagen matrices were imaged using an 8 megapixel digital camera and processed using ImageJ.
2.7.4 Adipogenic induction
70-‐90% confluent cells were cultured in adipogenic induction medium (αMEM supplemented with 10% FBS, 1% P-‐S, 1% GlutaMax, 0.1nM dexamethasone, 0.45mM IBMX, 10ug insulin and 125uM indomethacin) for 28 days. Control cells were
cultured in αMEM supplemented with 10% FBS, 1% P-‐S, 1% GlutaMax. After adipogenic induction, cells were fixed in 4% formaldehyde for 1h at 4oC followed by
gentle washing with MQ water. Fixed cells were then incubated in 2mls 60% isopropanol for 5 minutes followed by addition of 3 parts oil red O stock (3g/L oil red O in isopropanol, sterile filtered) and 2 parts MQ water for a further 5 mins. Cells were then carefully rinsed in MQ water until clear and imaged (in HBSS) using a Leica DMIL microscope with the acquisition software Leica Application Suite and processed using Adobe Photoshop (Fink and Zachar, 2011).
2.7.5 Osteogenic induction
70-‐90% confluent cells were cultured in osteo-‐base medium (OBM; αMEM
supplemented with 20% FBS, 1% PS, 2% GlutaMax, 5mM β-‐glycerophosphate and
50µg/ml ascorbate-‐2-‐phosphate) for 7 days followed by culture in osteo-‐
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21 days (control cells were cultured in αMEM supplemented with 20% FBS, 1% P-‐S, 2% GlutaMax). Cells were fixed for 1h at 4oC in 4% formaldehyde and calcium
deposits were stained with 40mM alizarin red (pH 4.1). Images were taken on a Leica DMIL microscope using the acquisition software Leica Application Suite and processed using Adobe Photoshop (Krause et al., 2011).