FN-silk protein (3 mg/mL) were placed as a defined drop (15–40 µL) spread out to a diameter of 1 cm in the middle of a well of a non-treated, hydrophobic 24-well plate (Sarstedt). Air was quickly pipetted into the drop 20–30 times. Cells (0.5–2 × 106/mL) suspended in the appropriate culture media (containing 25 mM Hepes, without serum), were added dropwise (10–20 µL) either before or after introduction of air bubbles. The foams were kept 15–60 minutes in the cell incubator before covered with complete cell culture medium. For foams integrated with hESC, 15 µl of FN-silk was used for 50 000 hESCs (15 000 cells/µl). Cells were integrated in the presence of 10 µg/ml Rock inhibitor Y27632 (VWR) and silk was stabilized for 15 min at 37 °C in 5% CO2. After stabilization, 0.7 ml NutriStem supplemented with 10 µg/ml Rock inhibitor Y27632 was added. Rock inhibitor was omitted in the medium from 24 hours after seeding. Larger foams were prepared from 1.5 mL of FN-silk (3 mg/mL) subjected to 5–10 sec of whipping using a small battery operated hand whisker (Rubicson).
Rock inhibitor y27632
Manufactured by Avantor
Rock inhibitor Y27632 is a small molecule that selectively inhibits the Rho-associated protein kinase (ROCK) enzymes. ROCK enzymes play a role in regulating cell shape and motility. Y27632 has been widely used in cell culture and research applications as a tool compound to study ROCK-dependent cellular processes.
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Lab products found in correlation
2 protocols using rock inhibitor y27632
1
Fabrication of FN-silk Protein Foams
2
Cultivation of hPSCs in Silk Foam
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Silk foam with integrated hPSCs were prepared in hydrophobic plates of 24-well format (Sarstedt cat no 83.3922.500). Porous foam was formed by rapidly pipetting air (2-3 strokes per s) using a pipette set at 40 µl into a droplet of 20 µl FN-silk/ LN-521 placed at the bottom of a well and spread to a diameter of 1 cm. 50 000 hPSCs resuspended in culture medium (typically 10 000-15 000 cells per µl) in presence of 10 µg ml -1 Rock inhibitor Y27632 (VWR) were added directly into the foam and distributed by 5 additional strokes with the pipette. The foams were stabilized for 15 min at 37 °C, 5% CO 2 in a cell incubator before submerged with 0.7 ml Essential 8™ (ThermoFisher) supplemented with 10 µg ml -1 Y27632. Rock inhibitor was omitted in the medium from 16-20 hours after seeding. For evaluation of foam stability, 12 foams per condition were monitored by bright field microscopy 2 days after foam formation. Stability was graded as % of intact foam attached to the bottom of the well. After the air-bubbles have burst, the foam maintains at least 100 µm height, which, if assuming around 5 cell layers, a similar cell density as the 2D control (14 × 10 3 cells per cm 2 ).
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