19. Valuable insight into the distribution of the cells on the MCs,
cell confluency on single MCs and MC aggregate size can be
gleaned by fixing the MC bound cells with 10% neutral buff-
ered formalin solution (Sigma-Aldrich), staining the cells with
DAPI, and analyzing the MCs using the EVOS® FL Auto
Imaging System (Thermo Fisher Scientific). The staining pro-
cedure results in a shift in the excitation (λex) and emission
(λem) wavelength from 340 to 364 nm and 488 to 454 nm,
respectively, when DAPI selectively binds to double-stranded
DNA. This causes the cell nuclei to fluoresce. Thus, when
images taken using both the phase contrast and DAPI filter
lens at 4 magnification are merged, a single image showing
both the orientation of the MCs, as well as the location of
individual cell nuclei emerges. These images may then be
used to draw conclusions regarding cell distribution and MC
aggregation.
20. DAPI working solution is prepared by dissolving DAPI
(Roche) in WFI to a final concertation of 1.43 mmol L1.
This solution is light and temperature sensitive and should
therefore be stored in an opaque container at either 2 to 8 C
for up to 6 months or 15 to 25 C for up to 12 months.
21. Prepare a 0.3% Triton® X-100 working solution by diluting
30 μL of Triton® X-100 (Sigma-Aldrich) in 10 mL of sterile
DPBS. The working solution may be further improved
through sterile filtration (using a 0.22-μm syringe filter)
which removes any solid impurities introduced by the deter-
gent, as these may impact the quality of the images taken post-
staining.
22. Staining buffer is a mixture consisting of 19 mL autoMACS®
Rinsing Solution (Miltenyi Biotec) and 1 mL MACS BSA
Stock Solution (Miltenyi Biotec). The buffer contains sodium
azide as a preservative, serum to minimize nonspecific binding
of antibodies to the cells and ethylenediaminetetraacetic acid to
reduce cell aggregation.
Acknowledgments
The authors would like to thank Joel R€ath and Riccardo Pianezza,
as well as Dr. Tiziano Tallone for their assistance and support with
the cell cultivation experiments. Finally, some figures were rendered
using the intuitive online application BioRender (see www.bio
render.com for more details).
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Misha Teale et al.