293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and penicillin/streptomycin. Human neonatal epidermal keratinocytes (HK; Lonza Cat# 00192906) were cultured in EpiLife medium (Thermo Fisher Scientific). SCC4 (ATCC Cat# CRL-1624; RRID: CVCL_1684), SCC9 (ATCC Cat# CRL-1629; RRID: CVCL_1685), SCC15 (ATCC Cat# CRL-1623; RRID: CVCL_1681), and SCC25 (ATCC Cat# CRL-1628; RRID: CVCL_1682) cells were obtained from the American Type Culture Collection (ATCC) and were cultured in DMEM/F12 (Mediatech, Corning Life Sciences) containing 10% FBS, penicillin/streptomycin, and 400 ng/mL hydrocortisone (Sigma-Aldrich). Cell morphology and confluence assessments were performed using an IncuCyte FLR live cell imaging system (Essen BioScience).
Incucyte flr live cell imaging system
Manufactured by Sartorius
Sourced in United States
The IncuCyte FLR live cell imaging system is a real-time, automated cell imaging platform designed for continuous monitoring of live cell cultures. The system provides high-quality, quantitative data on cell proliferation, morphology, and other cellular changes over time, without the need for manual intervention.
Automatically generated - may contain errors
Lab products found in correlation
Cellplayer software module, by Sartorius (2 mentions)
96 pin woundmaker, by Sartorius (2 mentions)
Imagelock plate, by Sartorius (2 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Biocoat invasion chamber, by BD (1 mentions)
96 well woundmaker, by Sartorius (1 mentions)
Countess cell counter, by Thermo Fisher Scientific (1 mentions)
Celltiter aqueous one solution reagent, by Promega (1 mentions)
96 well plate, by Corning (1 mentions)
Woundmaker, by Sartorius (1 mentions)
Incucyte software, by Sartorius (1 mentions)
Essen imagelock, by Sartorius (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
9 protocols using incucyte flr live cell imaging system
1
Cell Culture Conditions and Assessment
2
3D Matrigel Invasion and Proliferation Assay
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3D Laminin-rich Extracellular Matrix (lrECM) On-Top Cultures (referred to as 3D On-top Matrigel assays) were performed with an initial seeding density of 1x103 PC3 cells per well of a 96-well plate and conducted as previously.45 (link) For invasion assays, 15 000 PC3 or 25 000 DU145 cells were seeded overnight into Matrigel-coated (100 μg/ml in growth media) wells in a 96-well Image-lock plate (Essen BioScience Inc., Ann Arbor, MI, USA). Wounds were made through the monolayer of confluent cells using the 96-pin WoundMaker (Essen BioScience Inc.) according to the manufacturer’s instructions. Wells were washed twice with phosphate-buffered saline and matrigel (50 μl, 1 mg/ml in growth media) was added to each well and allowed to solidify before the initiation of imaging. Images were captured every 2 h for up to 48 h by the IncuCyte FLR live cell imaging system (Essen BioSciences Inc.). Wound closure kinetics were determined using the CellPlayer software module (Essen BioScience Inc.). For proliferation assays, cells were seeded as described for invasion assays and cell confluence determined using the same software. Proliferation was also assessed using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific).
3
Cell Proliferation Quantification via IncuCyte FLR Imaging
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For measurement of cell proliferation the IncuCyte FLR live cell imaging system (Essen BioScience, MI, USA) was used to quantify the confluency of living cells. Calculation of the doubling times (Td) was done using the online calculator at http://www.doubling-time.com/compute.php (equation used: amount = 0.5288×e0.0317×time).
4
Cell Migration and Invasion Assays
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Seventy-five thousand cells were seeded in triplicate into 24-well cell culture inserts (8 µm pore, BD Biosciences) for migration assays or into BD BioCoat invasion chambers (BD Biosciences) for invasion assays. As chemoattractant, 10% FBS was added in the bottom chamber. After 24 hours, cells on the upper filter surface were removed with a cotton swab, while those on the lower surface were fixed and stained using the Diff-Quick staining kit (Lab Aids). Cell migration or invasion was quantified by counting eight random fields per filter using a light microscope (Olympus BX51). To assess proliferation, 35000 cells were seeded in quadruplicate into 24-well plates and assayed for cell density at 4 hour intervals over 72 hours using the IncuCyte™FLR live-cell imaging system (Essen BioScience). The data was analysed using the IncuCyte™ cell proliferation assay algorithm.
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Melanoma Cell Migration Assay
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Measuring A549 Lung Tumor Cell Viability
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Proliferation and Migration Assays for Prostate Cancer Cells
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For proliferation assays, 5000 LNCaP cells or 3000 PC3 cells were seeded overnight into 96‐well plates (Corning, In vitro Technologies, Eight Mile Plains, Australia). Cells were washed in PBS, and fresh medium containing 1% FBS or 1% CSS was added. After 72 h, proliferation was assessed using CellTiter AQueous One Solution Reagent (Promega, Hawthorn, Australia). For migration assays, 20 000 LNCaP cells or 15 000 PC‐3 cells were seeded in a 96‐well Image‐lock plate (Essen BioScience Inc., Ann Arbor, MI, USA). Wounds were made through the monolayer of confluent cells using the 96‐pin WoundMaker (Essen BioScience Inc.) according to the manufacturer’s instructions. Wells were washed twice with PBS and fresh medium (RPMI + 1% FBS) was added before the initiation of imaging. Images were captured every 2 h for up to 72 h by the IncuCyte FLR live cell imaging system (Essen BioSciences Inc.). Wound closure kinetics were determined using the CellPlayer software module (Essen BioScience Inc.).
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Real-Time Cellular Confluency Monitoring
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Wound Healing Assay with Mitomycin C
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Cells were seeded in gelatin coated 96-well plates (Essen ImageLock, Essen Bioscience) at 80% confluence and incubated overnight to allow them to reach confluence. Cells were treated for 2 hr before wounding with 2 μg/ml mitomycin C (M4287, Sigma) to block cell proliferation and scratches were performed in the cell monolayer using the wound maker (Essen Bioscience). Cells were washed immediately three times with PBS and re-fed with growth medium. Cell migration was monitored with an Incucyte FLR Live-Cell imaging system equipped with a 20X objective (Essen Bioscience). Images were acquired every 3 hr for 48 hr. Migration was quantified by measuring the relative wound density of at least three biological replicates in each experiment, using the Incucyte software (Essen Bioscience) as recommended by the manufacturer.
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