Download Special Issue – Exosomes Colon metastasis exosomes

Special Issue – Exosomes
Colon metastasis exosomes
Supplemental Material
Materials and methods for Supplemental Figure 1
Growth assay for estimation of proliferation rate
SW480 and SW620 cells in RPMI-1640 medium supplemented with 10% FCS, 60 µg/mL
benzylpenicillin and 100 g/mL streptomycin at 37C and 10% CO2 atmosphere, were
seeded in a 24-well plate at a density of 5.0 × 104 cells/mL and incubated at 37 °C, 10% (v/v)
CO2. Over ten days, cells from a single well were washed twice with PBS, lifted with 0.1%
(v/v) trypsin-versene and counted with a haemocytometer. The average of four fields on the
haemocytometer was calculated. The assay was repeated in triplicate.
Wound-healing assay
Wound-healing assays were conducted in 6-well plates. Cells were cultured to confluency.
Several light scratches were made radially with a plastic pipette tip (‘wounding’). The cells
were washed carefully 5× with warmed RPMI-1640 media and phase contrast images were
viewed with an inverted microscope (Eclipse TE300; Nikon) equipped with a 10× objective
(Plan Fluor; Nikon). Cells were photographed with an attached 12.6 megapixel digital camera
(DXM1200C; Nikon) and images processed with Nikon Elements Imaging Software (v3.0,
SP4). Cells were then incubated at 37 °C, 10% (v/v) CO2. At 24 and 48 h time points, cells
were washed and photographed as described. Cell migration was assessed based on the extent
of closure of the wound, as well as the pattern of closure. Assays were repeated in triplicate
for each cell line.
Cell-matrix adhesion assay
A solution of rat tail collagen type I (2 mg/mL, BD Biosciences) containing 0.2% (v/v) acetic
acid in ddH2O was passed through a 0.45 μm Supor membrane filter (Pall). This solution was
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Special Issue – Exosomes
Colon metastasis exosomes
diluted 1:5 in ddH2O and 400 μL added to wells of a 24-well plate. The plate was then placed
in a tissue culture hood overnight under UV. The same procedure was followed using BSA
(instead of collagen). Cells were seeded at a density of 5.0 × 105 cells/mL in RPMI, 0.5%
(w/v) BSA, 2 mM CaCl2, 2 mM MgCl2, and incubated for 60 min at 37 °C, 10% (v/v) CO2.
Cells were washed five times with HT PBS, 2 mM CaCl2, 2 mM MgCl2, and fixed for 20 min
in 5% (v/v) glutaraldehyde. Cells were subsequently washed and stained with 0.1% (w/v)
crystal violet, 200 mM MES, pH 6 for 10 min. Cells were washed five times in ddH2O and
allowed to dry, before being photographed under phase contrast conditions as described in
Wound-healing assay section. Finally, cells were solubilised via addition of 1 mL 10% (v/v)
acetic acid and placed for 10 min on an orbital shaker. Three aliquots of each solubilised cell
line incubated on collagen or BSA was placed into the wells of a 96-well plate and the
absorbance at 560 nm (assay) and 690 nm (background absorbance wavelength of plate)
measured. The assay absorbance was then subtracted from the background absorbance in
order to determine the degree of matrix adhesion.
Transwell assay
Six hundred microliters of RPMI-1640, 10% (v/v) FCS was added to the wells of a 24-well
plate and Transwell® inserts (growth area 0.33 cm2, pore size 8 μm) (Corning) submerged in
this media for 1 h to pre-condition membranes. The inserts were then relocated to a fresh 24well plate containing 600 μL RPMI 1640 media, 10% (v/v) FCS. Cells were seeded at a
density of 1.0 × 106 cells/mL in serum-free RPMI 1640 media, 0.1% (w/v) BSA within the
inserts and mixed well to disperse evenly over the membrane. After 24 h incubation at 37 °C,
10% (v/v) CO2, the inserts were washed twice with PBS and the cells fixed with methanol for
2 min. The cells were washed twice with PBS and stained with haematoxylin for 30 min.
Cells residing on the inner portion of the insert were removed with a cotton bud, and ten
random phase contrast images per well obtained as above. The number of cells per image was
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Special Issue – Exosomes
Colon metastasis exosomes
then counted and the average count used to assess cell migration. The assay was repeated in
triplicate for each cell line.
Soft agar growth assay for colony formation
Basal agar was prepared by mixing even parts of 1.2% (w/v) agar in sterilised ddH20, and 2×
RPMI 1640 media, 20% (v/v) FCS. 150 L of this mixture was added to 35 mm dishes and
allowed to set at RT for 30 min. Cells were then layered over the basal agar at a density of
5000 or 10,000 cells/dish in growth media + 0.3% (w/v) agarose. This layer was allowed to
set for 30 min. Cells were cultured for at 37 °C, 10% CO2 (v/v) for 15 days, with media
refreshment 3 times/week. At the conclusion of this period, cells were fixed and stained with
crystal violet as per the cell-matrix adhesion assay and phase contrast images taken as
described.
Invasion assay
One hundred microliters of 1 mg/mL rat tail collagen type I (BD Biosciences), 10% (w/v)
matrigel (BD Biosciences) in RPMI-1640 was coated on each well of a 24-well plate and
incubated at 37 °C, 10% (v/v) CO2 for 4 h to solidify. Cells were suspended at a density of
3.33 × 105 cell/mL in fresh collagen/matrigel mixed at the above ratio. 150 L of the
suspension was quickly applied to each well. After 4 h incubation at 37 °C, 100 μL RPMI
1640 media was added to each well and cells checked daily for growth behaviour in the
collagen/matrigel. The RPMI media was changed daily. After 1 week, cells were imaged
under phase-contrast microscopy conditions. The degree of invasiveness was assessed by
examination of cell spreading at the bottom and middle of the collagen/matrigel mixture.
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