35 mm glass bottom petri dish

To prepare substrates for cell migration study, a small drop of Sylgard^® 184 poly(dimethyl siloxane) (PDMS) mixture was added to the center of the glass slide of a 35-mm glass bottom petri dish (MatTek, Ashland, MA). The mixture was cured at room temperature for 24 h to create a non-adhesive area in the center. A 3-mL cell suspension was added to the petri dish and cells were allowed to attach overnight. A single hydrogel microfiber collected on a stainless steel metal frame (diameter: 10 mm, thickness: 1 mm) was washed with PBS and cell culture media three times each, followed by exposure to germicidal UV light for 15 min. Subsequently, the fiber fixed by the frame was laid on top of cells across the PDMS center. Cells were incubated overnight in the presence of the immobilized microfiber and live cell imaging was performed using a Zeiss LSM 880. Z-stack and tile images were collected every 15 min over a 12-h period. Images were processed using Zen software (Carl Zeiss, Thornwood, NY) and videos were created with ImageJ. Latrunculin B (Lan B) and nocodazole were added in situ to a final concentration of 10 μM for F-actin and microtubule inhibition, respectively. After the inhibitor solutions were added, live cell confocal imaging was carried out for 3 h.

We placed a 20 µL drop of proteoliposome suspension in the center of a 35 mm glass-bottom Petri dish (MatTek, Ashland, MA) that we had pretreated with 5% milk (w/v dry nonfat milk powder in phosphate buffered saline) for 30 min. Then we covered the dish with 2 mL of bath solution while taking care to keep the giant proteoliposomes near the center of the dish. Most of the giant proteopliposomes settled to the bottom of the dish within a few minutes. The bath solution consisted of 130 mM KCl, 1 mM MgCl₂, and 10 mM HEPES with a pH titrated to 7.2 using KOH. The pipette solution was identical to the bath solution supplemented with 5 mM ATP. We fabricated the patch electrodes with resistances of 5.0–10.0 MΩ from borosilicate glass (Sutter Instruments, Novato, CA) using a P-87 puller (Sutter Instruments, Novato, CA). After formation of a Giga seal, we pulled the patch electrode away from the giant proteoliposome and quickly went through the water–air interface in order to ensure the inside-out configuration. We recorded the single channel current at room temperature using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) and digitized the recordings using a Digidata 1322A (Molecular Devices, Sunnyvale, CA). The cut-off filter frequency was 5 kHz and the sampling rate was 25 kHz. Data acquisition was done using pClamp9 software (Molecular Devices, Sunnyvale, CA).