Setup and user guide

1
ˆ¶  ˝u
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Æ££
ª¢`‡.1
‡—'ª›'.2
Æ££.3
‰ª.4
.5
›»Æ`›§ª›.6
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˚§'¸.7
4`¿›.8
fl
ˇ›˚§Æfl‡—Æ'§‡
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. ›¯Æ§
ø—•›fl .1
›»`¿'¸
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15°›fl.3
›¿¸¯£›fl.4
‡—
›ª'¸‡ª›fl.5
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`—¿£¢ª
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ªø—•
•• .12
ª›ª.13
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single piece of film. For each exposure condition, findings representative of the range of
FX detected are shown. (B) FXa was detected in airway casts from CEES-exposed lungs.
Rat FXa migrates at 37 kDa and was confir med by comparison to pur ified preparations
that are shown in the three lanes at far right .
Figure 5. (A) Cytotoxicity of in vitro exposur e to CEES. 16HBE cultures were exposed to
diluent (DMSO alone) or dilu ent containing increasing con centrations of CEES (500,
750, 1000, or 1500 µ M). After 24 h, cell viability was determined by MTT assay. Values
represent mean ± SD of three independe nt experiments. *denotes statistical difference
from diluent exposure (0 µ M CEES). (B) Effect of CEES exposure on caspase 3/7
activation. 16HBE cells were exposed to either diluent (DMS O alone) or diluent
containing CEES (750 µ M) for 6, 12, or 18 h. At each time point, cultures were separated
into samples consisting of the adherent cell fraction (light column) or the media fraction
(dark columns) and tested for caspase 3/7 activity. Each column represents average
values from 9 replicate cultures exposed to CEES in 3 independent experiments. *p<0.05
as compared to diluent (DMSO) control.
Figure 6. Effect of CEES exposure on clotting time of plasma induced by culture media
obtained from 16HBE cells. Cells were e xposed to diluent (DMS O alone) or diluent
containing CEES (750 µ M) for 6, 12, or 18 h. Small volumes of media were then used in
a “time to clot” assay by addition to naïve plasma in the presence of CaCl
2
. (A)
Histograms demonstrating the time-dependent increase in absorbance at 405 nm, which
occurs as a function of fibrin clot forma tion. (B) Columns represent the mean clotting
40
Figure 9. Total cellular expression of TF fo llowing CEES exposure. (A) Untreated or
CEES-exposed (750 µ M) 16HBE cells were harvested af ter 4, 8 or 18 h of exposure and
analyzed for TF abundance by Western blot. Expression of  -actin was measured to
confirm that comparable levels of protein were loaded from all samples. The blot is from
a representative experiment (n=3 repetitions). (B) Columns represent the mean density +-
SD (arbitrary units) of 3 se parate blots after quantification by imaging densitometry and
normalization to Š actin. No statistical difference was observed between groups as
assessed by ANOVA. ( C) Effect of CEES exposure on the release of TF. 16 HBE cell
cultures were exposed to DMSO or 750 uM CEES for 18 h and separated into an
adherent cell (black bar) fraction, a fraction of pelleted floating cells from media (white
bar), or microparticles (grey bar). Material in each fraction was washed and resuspended
at equal volume and assayed for TF as desc ribed in “Methods”. Data are the means +/-
SD for 4 determinations. (* P<0.05 comp ared to corresponding DMSO control).
Figure 10. Impact of lactadherin and recombin ant tissue factor path way inhibitor (rTFPI;
tifacogin) on the procoagulant activity of 16HBE cell media following CEES exposure.
(A) Cell media obtained 18 h after CEES exposure was incubated with varying
concentrations of bovine serum albumin (BSA), lactadherin or tifaco gin and tested in a
“time to clot” assay by its addition to naïve plasma. Each column represents the mean
clotting time in seconds ± SD for each treatment, as well as plasma treated with saline
only or culture media (18 h post-CEES expos ure) not incubated with anticoagulants.
“Time to clot” was quantified by determining the time at which absorbance values
plateaued. *p<0.05 and denotes significant di fference from the CEES-exposed cell media