Incucyte 96 well imagelock microplate wells

IncuCyte 96-well ImageLock Microplate wells (Sartorius, Göttingen, Germany) were coated with 300 μg/mL Myogel for migration and invasion assays (31 (link)). The cells were seeded at a density of 25,000 cells per well in 100 μL of complete medium for both assays. After 24 hours at 37°C, a 96-pin IncuCyte WoundMaker Tool (Sartorius) was used to make uniform wounds on the confluent monolayer of the cell. The wells were washed two times with media, and 100 μL of complete medium was added. For the invasion plate, 50 μL of Myogel-collagen gel (2.4 mg/mL Myogel, 0.8 mg/mL type I rat tail collagen) (Corning Incorporated, Corning, NY, USA) was added on top of the cells. After the gel was solidified, 50 μL of media was added, and the plates were transferred to an incubator. The wound closing was monitored automatically every 2 hours for two days using IncuCyte S3 Live-Cell Imaging System (Sartorius). Analysis of wound closing (width of the wound) was performed using Matlab. Mathematical function decorrelation was used to make the cells’ intensity substantially higher than the background.

IncuCyte 96-well ImageLock Microplate wells (Sartorius, Göttingen, Germany) were coated with 300 µg/ml Myogel, p-LG or h-LG for migration and invasion assays and incubated at 37 °C in a cell culture incubator overnight. The excess of the gels was removed before seeding the cells. For both assays, the cells were seeded at a density of 25,000 cells per well in 100 µl of complete medium. After 24 h at 37 °C, a 96-pin IncuCyte WoundMaker Tool (Sartorius) was used to make uniform wounds on the confluent monolayer of the cells. The wells were washed two times with medium, and 100 µl of complete medium was added. For the invasion assay, 50 µl of Myogel-collagen or Lymphogel-collagen matrix (2.4 mg/ml Myogel/p-LG/h-LG and 0.8 mg/ml type I rat tail collagen) (Corning) was added on top of the cells. After the matrix was solidified, 50 µl of complete media was added, and the plates were transferred to an incubator. The wound closing was monitored automatically every 3 h for 24 h using IncuCyte S3 Live-Cell Imaging System (Sartorius). Analysis of wound closing (width of the wound) was performed using Ilastik (freeware) [24 (link)] and Fiji software [23 (link)]. Results represent the average of three independent experiments, performed in quadruplicates.