Cells were transfected with siRNA against human CD44 (SI00299705 FlexiTube
siRNA Premix, Quiagen) for 72 h at 25 nM, after which cell monolayers
(ca. 5 × 105 cells) were incubated with 0.17 nM
nanoparticle-spiked PBS (Ca2+/Mg2+) for 3 h,
washed with PBS, dissociated in 0.2% EDTA (PBS), pelleted, and labeled with 20
μL of FITC-anti-human CD44 (clone MEM-85; Life Technologies)
for 30 min at 4 °C. Cells were recovered by centrifugation (5 min, 600 rcf),
fixed in 500 μL of freshly prepared 3.7%
paraformaldehyde, and analyzed using a BD LSR II flow cytometer (anti-CD44-FITC: 488 nm
ex; 530/30 nm em; nanoparticles: 561 nm ex; 610/20 nm em).
siRNA Premix, Quiagen) for 72 h at 25 nM, after which cell monolayers
(ca. 5 × 105 cells) were incubated with 0.17 nM
nanoparticle-spiked PBS (Ca2+/Mg2+) for 3 h,
washed with PBS, dissociated in 0.2% EDTA (PBS), pelleted, and labeled with 20
μL of FITC-anti-human CD44 (clone MEM-85; Life Technologies)
for 30 min at 4 °C. Cells were recovered by centrifugation (5 min, 600 rcf),
fixed in 500 μL of freshly prepared 3.7%
paraformaldehyde, and analyzed using a BD LSR II flow cytometer (anti-CD44-FITC: 488 nm
ex; 530/30 nm em; nanoparticles: 561 nm ex; 610/20 nm em).