Download Supplementary Methods Quantification of Fg adsorption by

Supplementary Methods
Quantification of Fg adsorption by radiolabelling
Adsorption of Fg to Ch films was estimated using Fg labelling with the radioisotype 125I. Ch films
were prepared as described in the methods section and Fg was labelled with 125I (Perkin Elmer)
using the Iodogen Method (1). After labeling, 125I-Fg was purified in prepacked PD-10 Sephadex G25 M columns (Amersham Pharmacia) previously equilibrated in PBS. The yield of iodination was
approximately 95%, as estimated by trichloroacetic acid precipitation (TCA, 10% v/v). 125I-Fg
adsorption was performed using Fg solutions at 100g/mL with a final activity of 80cpm/ng, for
2hrs at RT. Afterwards, each sample was transferred to different radioimmune assays’ (RIA) tubes,
rinsed three times with PBS and maintained in PBS up to 7 days at 4ºC. Samples and Fg standard
solutions were counted in a -counter and the amount of adsorbed Fg determined as:
Fg (ng/cm2) = counts (cpm) / (AFg (cpm/ng) x S (cm2))
in which counts is the samples-associated radioactivity, AFg the specific radioactivity of Fg solution
and S is the area of the surface. Eight replicas were used to determine the number of counts. Tests
were carried out in the presence of 10mM NaI to suppress the adsorption of residual free 125I.
Isolation of peripheral blood human NK cells, T cells and macrophages
Peripheral Blood Mononuclear Cells (PBMCs) suspension was prepared by density gradient
centrifugation: buffy coats were diluted by adding an equal volume of PBS; then 2 volumes of the
diluted buffy coat were carefully added on top of one volume of LymphoprepTM (Axis-Shield),
without mixing; tubes were centrifuged at 800g for 30min at 23ºC and with no brake. PBMCs were
collected from the interface and were washed with cold PBS by centrifuging at 300g for 10min at
4ºC for as many times as necessary to remove platelets.
NK cells were purified by negative selection using the EasySep® human NK cell enrichment kit
(StemCell Technologies), according to manufacturer’s instructions. Briefly, PBMCs were
resuspended at 5×106cells/mL in 0.5% BSA/2mM EDTA/PBS in a 5mL polystyrene tube. Then, the
EasySep® Human NK cell Enrichment cocktail was added at a concentration of 50µL/mL of cells.
Cells were mixed and incubated at room temperature for 10min, magnetic particles were added at
100µL/mL cells and incubated at room temperature for 5min. Then, 0.5%BSA/2mM EDTA/PBS was
added to a total volume of 2.5mL, and the suspension was gently mixed. The tube was placed into
a purple EasySep® magnet and set aside for 2.5min. The magnet and tube were then inverted in
one continuous motion, pouring off the desired fraction into a new tube. In order to recover more
cells, the magnet was returned to an upright position, 2.5mL of 0.5%BSA/2mM EDTA/PBS were
added, cells were mixed and set aside for another 2.5min and the new fraction was collected to
the same tube.
NK cells purity was evaluated by analysis of CD56 and CD3 expression and purified NK cells were
kept at 37ºC/5%CO2 in NK cell medium (DMEM with L-glutamine (PAA) with 10% human serum
(type AB, PAA), 30% nutrient mixture F-12 (Ham, PAA), 50U/mL penicillin-streptomycin
(Invitrogen), 1x non essential amino acids (Invitrogen) and 1mM sodium pyruvate (Invitrogen)).
T cells were purified by negative selection using the RosetteSep® human T cell enrichment cocktail
(StemCell Technologies), according to manufacturer’s instructions: 50µl of enrichment cocktail
were added per ml of whole blood and incubated for 20min at room temperature. The sample was
then diluted with an equal volume of PBS/2%FBS and overlayed on top of the density medium
LymphoprepTM. Tubes were centrifuged for 20min at 1200g, at room temperature and without
brake. Finally, the enriched fraction of cells was collected from the plasma:Lymphoprep interface
and cells were washed twice with PBS/2%FBS. The percentage of isolated CD3+/CD56- cells was
higher than 95%.
Populations of Macrophages were differentiated from Monocyte-enriched populations obtained
from peripheral blood by adherence. Briefly, 50×106 PBMCs were incubated in tissue culture plates
for 0.5-2hrs at 37ºC/5%CO2 and non-adherent cells were washed away with warm PBS. Cells were
incubated and allowed to differentiate in α-MEM with 10%FBS (Lonza) and 50U/mL PenicillinStreptomycin (Invitrogen) for 7-10 days. Cells were collected upon treatment with PBS/5mM EDTA
or with Accutase (PAA) and platted in 24 well invasion plates in the day prior to performing the
invasion assay. Purity of Monocyte-derived Macrophages was assessed by examining cells
morphology by microscopy and analysing CD14 and HLA-DR expression by flow cytometry, and
was found to vary between 40 and 70%.
Phenotypic analysis
Phenotypic analysis of immune cells and MSC was performed by flow cytometry. Briefly, cells were
washed with 0.01% Azide/0.5% BSA /PBS and incubated in antibody diluted in 0.01% Azide/0.5%
BSA/ PBS for 30min on ice. Appropriate isotypes were used as controls. Cells were then washed
four times with cold PBS prior to flow cytometry analysis. MSC were fixed by incubating in 4%
paraformaldehyde (Sigma) prior to analysis. A total of 10 000 events were acquired in a Flow
Cytometer (FACSCalibur, Becton Dicksinson) and analyzed with FlowJo.
For MSC phenotypic characterization, the following mAbs were used: FITC-labelled anti-human
CD105 (clone MEM-226, used at 2:50µL), PE-labelled anti-human CD34 (clone 4H11, at 2:50µL),
and PE-labelled anti-human HLA-DR (clone MEM-12, at 2:50µL), all from Immunotools; PE-labelled
anti-human CD73 (clone AD2, at 5:50µL), PE-labelled anti-human CD90 (clone 5E10, at 5:50 µL),
both from BD Biosciences;
MSC differentiation assay
The cells potential to differentiate in adipocytes, chondrocytes or osteoblasts under appropriate
stimuli was tested as follows.
For adipogenesis, MSC were plated in 24 well plates until reaching confluence and were
stimulated with DMEM with low glucose and glutamax plus 10% FBS (PAA) and
penicillin/streptomycin and with or without the adipogenic supplements (dexamethasone (Sigma)
at 1x10-4M, IBMX (Sigma) at 0.5mM in 0.5M KOH (Merck), insulin (Sigma) at 10µg/mL in 0.02M HCl
(Sigma), indomethacin (Sigma) at 100M – adipogenic medium). After 2-3 days of culture,
adipogenic medium was replaced by insulin medium (DMEM/10%FBS/10µg/ml insulin). After 1-2
days medium was replaced by adipogenic medium and after 3 days again by insulin medium. Cells
were treated a third time with adipogenic medium and then maintained in insulin medium for one
week. To evaluate differentiation, cells were stained with Oil Red O: cells were fixed with 4%
paraformaldheyde for 30min at room temperature and stained with Oil red O for 20min at room
temperature. Cells were washed with distilled water twice and analyzed under an inverted
microscope (Zeiss Axiovert).
For chondrogenesis, 200 000 cells were incubated in 500µL of medium (high glucose (4.5 g/L)
DMEM supplemented with 50µg/mL ascorbic acid (Fluka), 40µg/mL L-proline (Sigma), 100µg/mL
sodium pyruvate, 100µg/mL ITS culture supplement (BD Biosciences) and penicillin/streptomycin)
without or with chondrogenic differentiation factors (0.1µM dexamethasone and 10ng/mL TGF-β3
(Immunotools)). Cells were placed in 15mL conical tubes, spun at 450g for 10min and incubated at
37ºC/5%CO2 for 28 days. Medium was replaced twice per week. Chondrogenesis was evaluated by
toluidine blue staining. Briefly, cell pellets were fixed with 4% paraformaldehyde, dehydrated with
a series of ethanol and embedded in paraffin. Sections of 5µm were cut, rehydrated and stained
for 5min with a 1% Toluidine blue (Fluka) / 1% sodium borate (Sigma) solution . Sections were
analyzed under an inverted microscope (Zeiss Axiovert).
For osteogenesis, cells were incubated for up to 21 or 28 days in DMEM with low glucose and with
glutamax supplemented with 10% FBS tested for osteogenesis (PAA) and with
penicillin/streptomycin, without (basal medium) or with added osteogenic supplements (100nM
dexametasone, 10mM β-glycerophosphate (Sigma), 0.05x10-3M ascorbic acid – osteogenic
medium). Medium was replaced twice per week. To evaluate differentiation cells were stained for
ALP activity and assessed for mineralization by Von Kossa staining. Cells were fixed with 4%
paraformaldehyde for 15min at room temperature and washed with PBS. For ALP staining, cells
were incubated in a Naphtol AS-MX phosphate solution containing the Fast Violet B salt (both
from Sigma) for 30min at 37ºC and washed with PBS. For Von Kossa staining, cells were washed
with distilled water and incubated in 2.5wt % silver nitrate (Sigma) for 30min under UV light,
followed by incubation in 5wt % sodium thiosulphate (Sigma) for 3min. Finally, cells were washed
and imaged under an inverted microscope (Zeiss Axiovert).
References
1.
1993 Iodine-125. Guide to radioiodination techniques. Little Chalfort: Amersham Life
Science.