Biomedical Engineering Reference
In-Depth Information
condition, in triplicates, per time point. For assaying
by ELISA, consider testing at least two time points.
For assaying by immunoblotting, consider testing at
least three time points. Leave some beads uncoated,
washed three times with PBS (
steps 2
-
5
below), to
be used as a negative control.
2. For coating: resuspend bead suspension in 1 ml PBS
per 100
l of suspension.
3. Centrifuge for 5 min in a microcentrifuge at 20,000 ×
g
,
4 °C. Remove supernatant carefully and discard it.
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4. Add 1 ml PBS and centrifuge as in
step 3
.
5. Repeat
step 4
once more.
6. Add LPS or poly(I:C) to a fi nal concentration of
100
g/ml in 1 ml of PBS.
7. Mix overnight at 4 °C with end-to-end mixing (a
rotating wheel is preferred).
8. Centrifuge at 20,000 ×
g
for 5 min at 4 °C in the
microcentrifuge and discard supernatant.
9. Repeat
step 4
three times.
10. Resuspend the pellet in the original volume of beads.
The coated beads are now ready to use.
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5.2. Detection of IL-6 and IL-12 secretion by ELISA.
1. On days 7-10 of BMDC culture, collect BMDCs using
PBS only and without trypsinization (
see
Note 4
) as
follows. Transfer the culture medium containing
BMDCs in suspension to a 50 ml conical tube.
Collect mildly adherent BMDCs by adding 5 ml of
ice cold PBS to each 10 cm dish and rigorous pipet-
ting, and transfer the resuspended cells in PBS into
the same 50 ml tube. Finally, add 8 ml of ice cold PBS
to each dish and incubate for 30 min at 4 °C.
Rigorously pipette again and transfer the fi nal sus-
pension into the same conical tube.
2. Centrifuge for 5 min, 150 ×
g
, at 4 °C.
3. Discard supernatant and resuspend BMDCs in
BMDC complete medium. Count BMDCs.
4. Seed BMDCs into 96-well round-bottom plates (1
well per time point) at 60,000 BMDCs/well in 50
l
of BMDC complete medium. Plate enough wells to
test the following conditions in triplicate: (1) unstim-
ulated (no beads added); (2) stimulated with
uncoated beads; (3) stimulated with LPS coated
beads; and (4) stimulated with poly(I:C) coated
beads. Inhibitors or vehicle control should be tested
under all stimulating conditions.
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