Biomedical Engineering Reference
In-Depth Information
5. Leave dishes on ice for 15-30 min to allow the beads to adhere
to the cells.
6. Incubate at 37 °C for 30 min to allow for phagocytosis.
7. Carefully remove media and wash dishes three times with
RPMI 1640 medium (without FBS) to remove non-phagocy-
tosed beads. Add BMDC complete medium for the 2 h chase.
8. Analyze by live cell imaging using an inverted fl uorescence
microscope (optimally a spinning disk confocal microscope)
equipped with an environmental chamber kept at 37 °C and
5 % CO
2
. If addition of CO
2
is not possible, add 20 mM
HEPES buffer (pH 7.4) to the cells immediately before
imaging.
See
Note 6
regarding imaging requirements. Figure
1
shows examples of single frames from movies taken at 1 frame/s
using an Olympus IX71 inverted spinning disk confocal micro-
scope equipped with a Hamamatsu ImagEM EMCCD camera
and LCI Chamlide stage-top incubation system for live cell
imaging. Movies were acquired over a 5-min time period using
MetaMorph software (Molecular Devices, LLC, Sunnyvale,
CA, USA). The image sequences were saved as TIF fi les, and
then further analyzed using Image J (NIH) as described below.
Fig. 1
Visualizing BMDC phagotubules and the effects of inhibitors of TLR signaling on their formation. BMDCs
from C57BL/6 mice were left untreated or were pretreated for 2 h with 5
M
BX795 (TBK-1 inh). Cells were then pulsed for 15 min with OVA-Texas Red coated beads and chased for 2.5 h,
all in the absence or presence of inhibitor as indicated. Cells were then imaged by spinning disk confocal
microscopy. Shown are still images of Texas Red fl uorescence (
top
) and corresponding DIC images (
bottom
).
Bars, 2.6
ʼ
M IRAK 1-4 inhibitor (inh) or 0.1
ʼ
ʼ
m
