EDTA device. A 4-μm high master was produced by spin-coating
SU8-2005 for 30 s at 5,000 rpm, pre-baking for 2 min at 95 °C and
UV exposure of 550 mJ/cm^2 through a PL-360-LP (Omega Optical)
optical filter on an EVG mask aligner 610 (EVG Group). The wafer was
post-exposure baked for 3 min at 95 °C, developed in SU8 developer
and then hard-baked at 150 °C for 5 min. The optical filter was necessary
for high-resolution features in SU8.
Multilayer devices. First, the 25-μm high control master was made by
spin-coating SU8-3025 (Microchem) for 30 s at 3,000 rpm. The wafer
was soft-baked for 15 min at 95 °C, then exposed to 200 mJ/cm^2 U V,
then post-exposure baked at 95 °C for 5 min, then developed in SU8
developer. Second, the fluid-layer master was first fully coated with
a submicron layer of GM1040-SU8 by spin-coating at 5,000 rpm for
2 min. The wafer was then baked at 95 °C for 5 min, exposed to 100 mJ/cm^2
UV, post-exposure baked at 95 °C for 30 min, and then developed
in SU8 developer. The 5-μm fluid-layer features were made on top
of the coated wafer by spin-coating SU-2005 for 30 s at 3,000 rpm,
soft-baking for 2 min, UV exposure of 550 mJ/cm^2 through the optical
filter, post-exposure baking for 3 min at 95 °C, and developing in SU8
developer. Third, the fluid-layer master was then prepared for the next
layer by spin-coating HMDS at 3,000 rpm for 30 s and baking for 1 min at
126 °C. Scotch Magic tape (3M) was applied over the alignment marks.
AZ-40XT-11D (MicroResist Technologies) was then coated at 3,000 rpm
for 30 s. The Scotch tape was then peeled off to reveal the alignment
marks. The wafer was then baked at 126 °C for 7 min, aligned and
exposed to UV at 500 mJ/cm^2. After a post-exposure bake for 10 min at
120 °C, it was developed in 726 MIF for about 5–10 min. The features
were then rounded to make parabolic channels at 130 °C for 2 min
(centre height of 26.6 μm).
Silanization. Before applying PDMS, wafers were placed in a desic-
cator with 10 μl of Trichloro(1H,1H,2H,2H-perfluorooctyl)-silane
(Sigma-Aldrich), a vacuum of 100 mbar was applied, and then the
desiccator was sealed for 1 h. One permanent coating was sufficient
for later uses.
PDMS device microfabrication
Microfabricated devices were generated as previously described^11 ,^13 ,^28 ,
with 1:10 PDMS and degassed for 2 min at 2,000 rpm (mix) and for
2 min at 2,200 rpm (defoam) in a mixer/defoamer (ARE-250, Thinky)
before use.
Confiner. PDMS (Sylgard 184, Ellsworth Adhesives) was gently poured
onto the wafers, then 10-mm coverslips were activated by plasma clean-
ing for 2 min at medium intensity (Harrick Plasma Cleaner, pdc-002,
Harrick Plasma) and pressed upside-down onto the PDMS-covered
wafers. The wafer was baked on a hot plate at 95 °C for 15 min, and the
300-μm width micropillar-coated coverslip was gently removed from
the wafer. A soft PDMS 10-mm diameter pillar (1:30) was poured into a
homemade metal mould, degassed under vacuum and baked at 80 °C
for 1 week.
EDTA device. Of 1:10 PDMS (Sylgard 184, Ellsworth Adhesives), 20–25 g
was poured onto the wafer contained in an aluminium mould in a Petri
dish, degassed in a vacuum desiccator and baked overnight at 80 °C.
The devices were then diced with a razor blade and 2-mm entry and exit
holes were punched (Harris Unicore biopsy puncher, Sigma-Aldrich).
Following punching, they were cleaned with tape, sonicated in ethanol,
blown dry and plasma-bonded to a coverslip.
Multilayer devices. PDMS (80 g; RTV615, Techsil) was used; 10 g of
PDMS was put on the control layer and spin-coated (500 rpm for 15 s
followed by 2,300 rpm for 60 s), and 70 g was applied on top of the flow
layer in a tight aluminium mould and further degassed in a vacuum
desiccator to pump out the remaining bubbles. Both layers were baked
at 80 °C for 45 min, and the flow layer was removed from the mould,
trimmed with a razor blade, and entry and exit holes were punched
with a homemade arbor press. Debris from both the flow device and the
control layer on its wafer were carefully removed with Scotch tape, the
two layers were plasma-cleaned (2 min at medium intensity), aligned
manually and bonded together. After overnight baking in 80 °C, the
chip was removed from the wafer, the control layer entry holes were
punched, and the chip was bonded to the glass coverslip by oxygen
plasma and kept at 80 °C until further use. Before the experiments, all
entry ports were connected with metal pins (NE-13-1003, New England
Small Tube Corporation).Migration assays
Before experiments, 2 × 10^5 T cells in 2 ml R10 medium were stained
with Hoechst 33342 for 30 min (1 drop, NucBlue, R37605, Invitrogen,
Thermo Fisher Scientific) when nucleus visualization was needed for
cell tracking.Collagen assay. A 3D collagen scaffold (final concentration of
1.7 mg/ml) was obtained by mixing bovine collagen (PureCol, Advanced
BioMatrix) in 1× minimum essential medium Eagle and 0.4% sodium
bicarbonate (both Sigma-Aldrich), with 3 × 10^5 cells in R10 medium at
a 2:1 ratio^5 ,^29. After casting the mix in homemade migration chambers,
gels were allowed to polymerize for 45 min at 37 °C, 5% CO 2. Migration
was observed by time-lapse video microscopy.Cell confiner assay. PDMS micropillars were placed on the soft pillar
on a homemade magnetic glass lid. When indicated, dish and confiner
were coated with 2 μg/ml of recombinant mouse ICAM-1/human Fc chi-
maera (796-IC, R&D Systems). The setup was placed in a dish containing
R10 medium in a humidified incubator at 37 °C, 5% CO 2 , 1 h before the
experiments. Cells (5 × 10^4 in 5 μl R10 medium) were then pipetted onto
the medium-freed micropillars and confined on a dish above a magnetic
ring, and R10 medium was added back in the dish ready for imaging.
For the myosin inhibition experiment, 50 μM para-nitroblebbistatin^30
was added to the medium (Optopharma).Primary T cell confinement assay. To measure actin flow speed and
cell curvatures, we used an agarose-confinement setup^8. In brief,
plasma-activated glass was overlaid with a 0.2 mg/ml solution of
poly(l-lysine)-graft-PEG copolymer (PLL(20)-g[3.5]-PEG(2); SuSoS) at
4 °C overnight. To form a 0.5% agarose block with 10% FBS (and standard
concentrations of other supplements), one part 2× HBSS buffer (Sigma)
and two parts RPMI supplemented with twice the concentrations of
all other supplements used in R10 medium were mixed together with
one part 1% high-molecular-weight agarose (850152, Biozym Scientific
GmbH) in water at 50 °C and subsequently cast onto the dish then al-
lowed to solidify at room temperature. CCL19 (250-27B, Peprotech) was
added to soluble agarose before casting. T cells were injected under the
agarose block with a micropipette and incubated for 30 min at 37 °C
under 5% CO 2 before imaging.EDTA migration assay. T cells or PLB cells (5 × 10^4 ) were intro-
duced into the 2-mm entry port of the microchamber, and the de-
vice was soaked in R10 medium. Cells were left to enter freely into
the confinement and channel zone for 1 h and then imaged with a
video microscope. After 1 h of imaging, chemical disruption of the
cell adhesions was performed by removing R10 medium from the
dish and adding R10 medium containing 10 mM EDTA (prepared
from 0.5M stock; EDS-100G, Sigma-Aldrich) with or without 50 μM
para-nitroblebbistatin^30 (Optopharma). Imaging was then resumed
for 1 h. For dendritic cells, experiments were performed 1 day after
overnight LPS activation (200 ng/ml; Sigma-Aldrich) with or without