Nunclon delta surface 6 well plates

The SCP1-HAS-eGFP and the SCP1-mock were incubated for 24 h in culture medium containing 10 mM GlcNAc and afterwards 24 h in serum-free culture medium containing 10 mM GlcNAc. For detachment of the cells, StemPro Accutase (Thermo Fisher Scientific) was used. The reaction was stopped with serum-free culture medium containing 10 mM GlcNAc and washed in the same medium. To perform the adhesion assay, 1.8 × 10⁴ cells were seeded on Nunclon Delta Surface 6-well plates (Nunc) in 4 mL. The adhesion was imaged with 60 frames/h for 150 min using an inverted microscope Axiovert S100 (Carl Zeiss, Oberkochen, Germany) with a 20× phase contrast objective equipped with a controllable biochamber (Pecon, Erbach, Germany). For every cell, the time point was determined when they formed the first visible protrusion differing from the round shape of unattached cells using FIJI software [42 (link)]. By applying a sigmoidal four-parameter-logistic in GraphPad Prism software (GraphPad Software, San Diego, CA, USA), the time point of 50% adherent cells was calculated. The experiment was performed three times with a mean value of 32 cells per line and repetition.

Cells were preincubated in culture medium containing 10 mM GlcNAc as described above. Control cells were treated with 500 U/mL hyaluronidase (HAse from bovine testes, Sigma-Aldrich) for 1 h at 37 °C and an additional washing step. Afterwards, cells were seeded on Nunclon Delta Surface 6-well plates (Nunc) in the following densities: 2000, 4000 and 10,000 cells/cm². 10, 20 and 40 min after seeding, the cells were washed two times with DPBS and fixed with 4% paraformaldehyde in DPBS. The 6-well plates were stained with BODIPY 581/591 SE (Invitrogen, Darmstadt, Germany), a dye binding to free amino groups via NHS esters. Per cell line, treatment and time point between 38 and 97 cells were imaged with an AxioObserver Z1 (Carl Zeiss) and their area was measured using the FIJI software [42 (link)].