Article
2.5 μg/ml CCL19 (250-27B, Peprotech) added on the opposite hole of
cell entry and with or without 30 mM EDTA^13.
Multilayered microfluidics: maze and microchannels migration
assay. As talin-KO cells failed to freely enter the confinement zone, a
homemade microfluidic setup was used to push the cells by applying
a pressure differential to the inlet and outlet ports of the device. The
microfluidics designs in Extended Data Fig. 2d were used, as well as
separated chambers containing the four pillar mazes (identical to
ref.^28 with four chambers). In brief, the core component of those
devices is the flow layer with a 5-μm height pillar maze (Fig. 2a–d,
Extended Data Fig. 2e) or channels (Figs. 2e–g, 4 , Extended Data
Figs. 2f, 4) with an exit, cell and medium entry ports and on one side
and on the other side a sink channel connected to two or three exits.
Flow and sink channels are rounded, 26 μm high. For a controlled
flow of cells and fluids, all ports are equipped with independently
controllable push-up PDMS membrane valves. In brief, the control
channels are connected to solenoid valves (MH1, miniature, Festo)
controlled with a MATLAB graphical user interface (MathWorks).
First, the chip was mounted into the 37 °C, 5% CO 2 chamber of the
microscope, and optimal closing pressures of 0.15–0.2 MPa of the
water-filler PDMS membrane valves were determined for each chip;
each valve was checked individually by microscopy. Second, the whole
chip was saturated with a pre-warmed medium by applying a pressure
of 0.04 MPa and incubated in R10 medium for 30 min. Cells were first
loaded via the ports on one side, resulting in their distribution along
the chamber, then slowly pushed with a pressure of <0.01 MPa from
the medium port towards the confinement zone, and further pushed
in the microchannels or micropillars maze. Finally, all valves were
closed to ensure that no external pressure or flow was applied to the
cells during imaging.
TIRF microfluidics: microchannels migration assay. For Fig. 4e–g,
TIRF microscopy was not possible with the multilayered microfluidic
devices, so devices with the flow layer only were generated. First,
R10 medium was forced into the device with a vacuum desiccator,
and then the chips were incubated in humidified 5% CO 2 at 37 °C for
2 h before the experiments. Experiments were performed on the
microscope stage (humidified environment 5% CO 2 at 37 °C) and a
low cell flow was delivered and stopped after talin-KO cell loading
in the microchannel using a LA120 syringe pump (5 μl/h; Landgraf
Laborsysteme).
Primary T cell confinement assay on nanoridges
T cells were confined under agarose as previously described with minor
modifications^8. In brief, plasma-activated coverslips with nanoridges
(800 nm/800 nm/600 nm (groove/ridge/depth)) (ANFS-CS25-50,
NanoSurface Biomedical) were passivated with a 1 mg/ml solution of
poly(l-lysine)-graft-PEG copolymer (PLL(20)-g[3.5]-PEG(2), SuSoS)
at 4 °C overnight. Flat control coverslips (FLAT-CS25-50, NanoSur-
face Biomedical) were either passivated as described above or coated
with rhICAM1-Fc (2 μg/ml) (720-IC-050, R&D) to generate adhesive
substrates. Next, 0.5% agarose was prepared by mixing A, 5 ml of 2×
HBSS buffer (Sigma), and mixing B, 10 ml of serum-free RPMI supple-
mented with 20% BSA and twice the concentrations of all other sup-
plements used in R10 medium (see above), and mixing C, 5 ml of 1%
high-molecular-weight agarose (850152, Biozym Scientific GmbH) in
PBS. Agarose solution (0.5%) was kept at 50 °C in a water bath before
it was layered on top of the coated coverslips and allowed to solidify
for 1 h at 4 °C. CCL19 (10 nM; 250-27B, Peprotech) was added to solu-
ble agarose before casting. T cells were isolated from the spleen of
wild-type and/or Lifeact-GFP mice (T cell isolation kit mouse; MACS,
Miltenyi Biotec) and kept in R10 medium at 37 °C and 5% CO 2. Before
imaging (30 min), 0.5 μl of highly concentrated T cells was injected
under the agarose block.
Time-lapse video microscopy
Bright-field video of T cells for collagen assays (×10 objective), 3-μm
high confiner assays and primary T cells migrating under agarose
(×20 objective) were acquired by time-lapse acquisition (time interval
of 20 s) using inverted cell culture microscopes (DM IL Led, Leica
Microsystems) equipped with cameras (ECO415MVGE, SVS-Vistek)
and custom-built climate chambers (5% CO 2 , 37 °C, humidified). A
5-μm high confiner, EDTA and microfluidics assays were recorded
every 30 s or 60 s at 2–6 multi-positions with NIS Elements software
(Nikon Instruments). Experiments were performed with an inverted
wide-field Nikon Eclipse Ti microscope in a humidified and heated
chamber at 37 °C and 5% CO 2 (Ibidi Gas Mixer), equipped with a
×20/0.5 NA PH1 air objective, a Hamamatsu EMCCD C9100 camera
and a Lumencor Spectra X light source (390 nm, 475 nm, 542/575 nm;
Lumencor). TIRF microscopy was performed with a ×60/1.46 NA oil
objective, optovar ×1 or ×1.6 in a humidified and heated chamber at
37 °C and 5% CO 2 using an inverted Axio Observer (Zeiss) microscope,
a TIRF 488/561-nm laser (Visitron Systems) and an Evolve EMCCD
camera (Photometrics) controlled by VisiView software (Visitron
Systems). To visualize actin retrograde flow, assays were recorded
every second for TIRF acquisition only (Fig. 1e–g, Supplementary
Video 3 example 3) or every 2 s or 3 s when acquisition of both TIRF
or wide-field fluorescence and bright field was necessary (Fig. 4e–g,
Extended Data Figs. 4b, 5g, Supplementary Videos 3 (examples 1 and
2), 10). When cells migrated outside the field of view (Supplementary
Video 10), they were manually followed via a joystick controller.
For the primary T cell confinement assay on nanoridges (Extended
Data Fig. 7), a video of actin flow (Lifeact-GFP) were recorded (2-s
frame rate) on an inverted spinning-disc confocal microscope
(Andor) using a ×100/1.4 NA objective and a 488-nm laser line in a
custom-built climate chamber (37 °C under 5% CO 2 ). For the primary
T cell flow analysis (Extended Data Fig. 8), inverted spinning-disc
confocal microscopy was performed at 37 °C under 5% CO2 with an
iMIC (TILL Photonics, FEI) instrument with a ×60/1.35 NA objective
and a 488-nm laser line.Image analysis
FIJI imaging processing software (https://fiji.sc/) was used for image
and video microscopy analysis.Spreading area. TIRF images of single cells were binarized after
de-speckling and background subtraction, and the spreading area
was measured using the Analyse Particles tool.Cell size measurement. To measure cell size, fluorescent images of
3-μm-confined control (Lifeact-mCherry) and talin-KO (Lifeact-GFP)
cells were binarized, and the single-cell spreading area and perimeter
were measured using the Analyse Particles tool.Manual tracking. A 1-h-long bright-field video of T cells in collagen
and 3-μm high confiners were reduced to a 1-min and 2-min interval,
respectively, and cells were tracked manually by using the ‘Manual
tracking’ plugin provided by Fiji.Automated tracking. For the 5-μm high confiner, pillar maze and
EDTA assays, cell migration was analysed by nucleus tracking using
TrackMate^31 (https://imagej.net/TrackMate). For the EDTA assay, tracks
before and after EDTA treatment were analysed and the speed was
extracted at the single-cell level in the different zones (2.5D confine-
ment, smooth and serrated microchannels containing only one cell).
For Fig. 4b, cell migration in the channel zones was regarded as efficient
for velocities above 0.5 μm/min. The mean square displacement was
calculated from the tracking files with a homemade script (MATLAB,
MathWorks).