Streptavidin solution

For live cell experiments, respective wells were incubated with 100 μl streptavidin solution (50 μg/ml; Sigma Aldrich) for 30 min at room temperature, followed by rigorous washing with PBS. Subsequently, biotinylated anti‐GFP antibody (10 μg/ml; antibodies‐online) was incubated for a further 30 min. Prior to cell seeding, wells were washed again with PBS. Cells were allowed to attach to the antibody‐patterned surface for at least 3–4 h. Total internal reflection fluorescence (TIRF) microscopy was carried out on an epi‐fluorescence microscope (Nikon Eclipse Ti2), where the samples were illuminated in TIR configuration (Nikon Ti‐LAPP) using a 60× oil immersion objective (NA = 1.49, APON 60XO TIRF). A multi‐laser engine (Toptica Photonics) was used for selective fluorescence excitation of CFP and YFP at 405 and 516 nm, respectively. After appropriate filtering using standard filter sets, the fluorescence was imaged onto a sCMOS camera (Zyla 4.2, Andor). The samples were mounted on an x‐y‐stage (CMR‐STG‐MHIX2‐motorized table, Märzhäuser), and scanning of the larger areas was supported by a laser‐guided automated Perfect Focus System (Nikon PFS). Quantitation of fluorescence contrast was carried out as previously.³⁸

The conjugation of biotinylated Sialyl Lewis^x (BSLeX) to the ADHLSCs’
surface through biotin–streptavidin bridges was performed in PBS at RT. ADHLSCs
were harvested with the different methods described earlier and washed with PBS.
The resulting cell pellet was dispersed in sulfonated BNHS solution (1 mM, 1
ml), and allowed to incubate for 10 min at RT. The cells were then washed with
PBS once to remove unattached and/or physically adsorbed BNHS from the cell
surface. A streptavidin solution (50 μg/ml in PBS, 1 ml) (Sigma-Aldrich) was
then used to treat the cells for 10 min at RT. The cells were washed with PBS. A
BSleX solution (5 μg/ml in PBS, 1 ml) (Glycotech, Gaithersburg, MD, USA) was
added to the streptavidin-conjugated cells, and the suspension was allowed to
incubate for 10 min at room temperature. Finally, the cells were washed with PBS
and resuspended in serum-free DMEM containing 4.5 g/l glucose (ThermoFisher
Scientific) with P/S (ThermoFisher Scientific). The concentration and viability
of the cells were evaluated by the trypan blue exclusion method.

A flow cell
with dimensions of 9 × 2 × 0.1 mm³ (L × W × H) was assembled
from two cover glasses 9 × 18 mm² and 40 × 50
mm² (MATSUNAMI) using a double-sided tape as a spacer.
First, the flow cell was filled with 5 μL of a 1 mg/mL streptavidin
solution (Sigma-Aldrich, S4762) and incubated for 5 min. The flow
cell was then washed with wash buffer (80 mM PIPES, 1 mM EGTA, 1 mM
MgCl₂, and ∼0.5 mg/mL casein; pH 6.8). Next, 5 μL
of a kinesin solution (800 nM) was introduced into the streptavidin-coated
flow cell. The flow cell was then incubated for 5 min to allow the
binding of kinesins to the glass surface through interaction with
streptavidin. After washing the flow cell with 10 μL of wash
buffer, 10 μL of an MT solution (200 nM, paclitaxel stabilized
GTP-MTs) was introduced and incubated for 5 min, which was followed
by washing with 10 μL of wash buffer. The motility of the MTs
was initiated by applying 5 μL of motility buffer containing
5 mM ATP. In the experiments where TMAO was used, 5 μL of motility
buffer containing 5 mM ATP and TMAO of prescribed concentrations was
infused into the flow cell. The MTs were monitored using a fluorescence
microscope within 5 min of ATP buffer addition. All of the experiments
were performed at room temperature (∼22 °C).