Biomedical Engineering Reference
In-Depth Information
5. Repeat
step 4
once more.
6. Activate the beads for conjugation by resuspending them in
0.5 ml of 8 % glutaraldehyde.
7. Mix for 4-6 h at room temperature on an end-to-end mixer
(preferably a rotating wheel, but a shaker will suffi ce).
8. Centrifuge for 5 min at 20,000 ×
g
and discard supernatant.
9. Repeat
step 4
once.
10. Add 1 mg/ml of fl uorescent OVA conjugate and 100
ʼ
g/ml
LPS in 0.5 ml PBS fi nal volume.
11. Mix overnight at 4 °C with end-to-end mixing (a rotating
wheel is preferred).
12. Centrifuge for 5 min at 20,000 ×
g
and discard supernatant.
13. Resuspend pellet in 1 ml of 0.5 M glycine and mix for 30 min
at room temperature. This step is performed to block unre-
acted sites on the beads.
14. Centrifuge for 5 min at 20,000 ×
g
and discard supernatant.
15. Repeat
step 4
twice. Fluorescent OVA is now covalently con-
jugated to the beads.
16. Resuspend the pellet in the original volume of beads. Fluo-
rescent OVA-coated beads can be stored for 1 week at 4 °C.
3.2 Phagocytosis
of OVA Fluorophore
Conjugated-Coated
Latex Beads
1. Differentiate and culture BMDCs in BMDC conditioned-
complete medium for 7-10 days on non-tissue-culture-treated
plastic dishes, as previously described (
see
Note 1
). For isola-
tion of mouse tissue-resident DCs
see
Note 3
.
2. On day 6 of BMDC culture, harvest BMDCs by washing the
dishes once with PBS and then treating them with trypsin-
EDTA for no longer than 5 min (
see
Note 4
). Collect detached
cells and recover by centrifugation for 5 min at 150 ×
g
(1,000 rpm in a Sorvall ST40R tabletop centrifuge with a
TX-750 rotor). Resuspend the cells at 200,000 BMDCs/2 ml
of BMDC conditioned-complete medium for each sample to
be analyzed. Seed 2 ml/dish in poly-
L
-Lys-coated 35 mm cul-
ture dishes. Seed one dish per condition per time point, to
allow for imaging at different time points.
3. On day 7, remove medium and add 1.5 ml of BMDC com-
plete medium without or with inhibitors. Add inhibitors either
2.5 h before the pulse, at the time of the pulse or at the start of
the chase at the concentrations indicated above (
see
Note 5
regarding testing the effi cacy and specifi city of the inhibitors).
Use DMSO at a comparable dilution as vehicle control.
4. Pulse the cells with beads. Carefully remove media from the
dishes and add 1.5 ml of BMDC complete medium (plus/
minus inhibitors) plus OVA-Texas Red and/or OVA-Alexa
Fluor 488-coated beads at a 1:200 dilution.
