For analysis of cell motility, HUVEC were transfected with control or raftlin siRNA. After 48 h, cells were seeded at low density to allow the observation of single cells. Cell motility was tracked over 14 h using an Incucyte ZOOM system (Essen Bioscience, Welwyn Garden City, UK). Cell velocity and directness of migration were calculated using Incucyte ZOOM software. For analysis of cell migration, HUVEC were transfected with control or raftlin siRNA. After 24 h, cells were seeded into a 96-well ImageLock plate (Essen Bioscience) and incubated for a further 24 h until confluent. Cells were serum-starved for 1 h and stimulated with 40 ng/ml VEGF. A scratch was made in each well using the IncuCyte WoundMaker tool (Essen Bioscience) and the plate was installed in the Incucyte ZOOM system. Images were taken at 10 × magnification every hour for 24 h. The percentage of wound closure was calculated using the Incucyte ZOOM software.
Incucyte zoom system
Manufactured by Sartorius
Sourced in United States, United Kingdom, Germany, Singapore, China
The IncuCyte ZOOM system is a real-time, live-cell imaging and analysis platform. It provides users with the ability to monitor and analyze cellular processes over time within a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Woundmaker, by Sartorius (13 mentions)
Incucyte zoom software, by Sartorius (12 mentions)
Incucyte zoom microscope software 2015a, by Sartorius (8 mentions)
Imagelock plate, by Sartorius (6 mentions)
Incucyte zoom, by Sartorius (6 mentions)
96 pin woundmaker, by Sartorius (5 mentions)
Matrigel, by Corning (4 mentions)
96 well plate, by Greiner (4 mentions)
Incucyte imagelock plate, by Sartorius (4 mentions)
96 well plate, by Corning (3 mentions)
Incucyte nuclight red lentivirus reagent, by Sartorius (3 mentions)
Incucyte woundmaker, by Sartorius (3 mentions)
Imagelock microplate, by Sartorius (3 mentions)
Celltox green dye, by Promega (3 mentions)
Incucyte cytotox red, by Sartorius (3 mentions)
Automated 96 wound maker, by Sartorius (3 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Prism 8, by GraphPad (3 mentions)
Incucyte caspase 3 7 apoptosis assay reagent, by Sartorius (2 mentions)
251 protocols using incucyte zoom system
1
Analyzing HUVEC Cell Motility and Migration
2
Cell Proliferation and Migration Assays
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Proliferation assays were performed by counting viable cells using a hemocytometer and by real-time quantitative live-cell proliferation analysis using IncuCyte® ZOOM system (Essen BioScience) as already described [26 ,39 (link)]. BrdU incorporation was measured also as described [26 ,39 (link)]. The scratch wound assays were performed by using IncuCyte® ZOOM system (Essen BioScience) and the cell migration assays were conducted in vitro in transwell chambers (Becton Dickinson) without additional migratory stimulants as already described [26 ,39 (link)].
3
Proliferation and Apoptosis Assays for Prostate Cancer
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For proliferation assays, PC3, DU145, TxS, TxR, cabR (5 000 cells per well), 22Rv1 TxS, TxR and cabR (10 000 cells per well) cells were seeded in 96-well plates (Corning) and incubated 37°C overnight. Cells were treated with different doses of BAY-1816032, docetaxel, cabazitaxel or their combination (in 10% CSS) and proliferation was measured by the Incucyte Zoom System (Essen Bioscience Inc., Ann Arbor, MI, USA). For apoptosis assays, at the time of treatment, cells were transfected with 1% (v/v) IncuCyte Caspase-3/-7 Apoptosis Assay Reagent (Essen Bioscience Inc.). Phase and green-fluorescent images were obtained at 10X every 4 h for 72 h and images were analyzed using the Incucyte Zoom software.
To assess mitosis duration, cells were seeded in 24-well plates at 20 000 cells per well and incubated at 37°C and imaged using the Incucyte Zoom System (4440, Essen BioScience Inc). Mitosis duration was evaluated as previously described.63 (link) Briefly, cells were imaged every 20 min for 48 h after the start of incubation at 20X magnification. Individual cells (50 cells per group) were tracked from the beginning to end of mitosis.
To assess mitosis duration, cells were seeded in 24-well plates at 20 000 cells per well and incubated at 37°C and imaged using the Incucyte Zoom System (4440, Essen BioScience Inc). Mitosis duration was evaluated as previously described.63 (link) Briefly, cells were imaged every 20 min for 48 h after the start of incubation at 20X magnification. Individual cells (50 cells per group) were tracked from the beginning to end of mitosis.
4
Tracking Cell Proliferation with IncuCyte
5
Tracking Cell Proliferation with IncuCyte
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All cell lines were transduced with IncuCyte NucLight Red lentivirus reagent (EF-1 Alpha promoter, puromycin selection) (Cat # 4476, Sartorius, Ann Arbor, MI, USA). Briefly, cells were seeded and allowed to adhere for 24 h to reach approximately 20%–40% confluence. NucLight reagent was diluted in DMEM containing 8 μg/mL polybrene (Cat # sc-134220, Santa Cruz Biotechnology, Dallas, TX) and added to cells for 48 h. Media was then replaced with fresh DMEM containing 3 μg/mL puromycin (Cat # MIR 5940, Mirus Bio, Madison, WI). Red fluorescence was monitored on the IncuCyte ZOOM system (Sartorius, Ann Arbor, MI), and analyzed using the IncuCyte image analysis software (Sartorius, Ann Arbor, MI). Similarly, shPHGDH cells were generated using lentiviral transduction particles (shPHGDH_1, TRCN0000028520, Sigma Aldrich, St. Louis, MO, USA; shPHGDH_2, TRCN0000233033, Sigma Aldrich, St. Louis, MO, USA). shGFP vectors were utilized as transduction controls. Knockdown of PHGDH was validated using the Wes Automated Western Blotting System (ProteinSimple, Bio-Techne, Minneapolis, MN), which uses automated capillary technology to measure protein expression and total protein concentration, per manufacturer’s protocol.
6
Clonal Expansion of hESCs from Single Cells
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Prior to dissociation hESCs (WA09) were stained with calcein-green AM (Thermo Fisher Scientific, C34852, 1:50,000) for 1h. Cells were then dissociated with Accutase (Thermo Fisher Scientific) for 10 min at 37°C. To ensure single-cell dissociation, the cell suspension was gently pipetted up and down 4 times and washed with PBS. Cells were pelleted, resuspended in StemFlex medium containing DMSO, Y-27632, or CEPT and filtered through 15 μm-cell strainers (pluriSelect). Cells were counted and resuspended at 5,000 cells/ml and loaded onto a microfluidic chip (Namocell). Single cells were dispensed using the Hana microfluidic single-cell dispenser (Namocell) onto LN521-coated 96-well plates containing StemFlex medium supplemented with DMSO, Y-27632, or CEPT. Side scatter, forward scatter and FITC gates were set to 25–400, 50–450 and 25–1000, respectively, to exclude doublets and ensure only live calcein green AM-positive cells were dispensed. The presence of single cells was confirmed using a Leica DMi8 microscope or IncuCyte Zoom system (Sartorius). Three days after single cells were dispensed a daily half-media change with StemFlex media was performed until day 10 post plating. On day 10, cells were stained with alkaline phosphatase (AP) stain II (Stemgent) and the number of wells with AP positive colonies were counted.
7
Quantifying Angiogenic Tube Formation
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The tube formation assay was employed to model the reorganization of angiogenesis. µ-Slides (8 well, ibidi GmbH, Gräfelfing, Germany) were coated with 10 µL Matrigel (Corning, New York, NY, USA). HUVEC were detached from the culture flask by trypsinization and resuspended in TKI-containing medium (controls were without TKI). Subsequently, 50 µL HUVEC cell suspension was transferred in triplicate to the Matrigel coated wells at a concentration of 10,000 cells/well and incubated in a humidified incubator at 37 °C und 5% CO2. Tube formation was monitored using the IncuCyte Zoom system (Sartorius, Göttingen, Germany) by taking images of each well every 30 min for 24 h. After 5 h incubation tube length was quantified with the software “WimTube Image Analysis” (Onimagin Technologies SCA, Cordoba, Spain).
8
Apoptosis Monitoring in Cancer Cells
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SNG-M, HCT116, A375, and SK-BR-3 cells were each seeded as per supernatant ELISAs and allowed to form monolayers overnight before addition of 0.5 μM (final) IncuCyte Caspase-3/7 Green Dye for Apoptosis (4440, Sartorius), either with or without 10,000 T cells per well. For the PDX assay, target cells were seeded as per supernatant ELISAs. The following day, ACL4 medium was washed out to R10 medium and 0.5 μM (final) IncuCyte Caspase-3/7 Green Dye for Apoptosis (4440, Sartorius), either with or without 80,000 T cells per well.
Plates were imaged from this point using the phase and green fluorescence channels in the IncuCyte ZOOM system (Sartorius) with 10× objective lens at 2-h (3 h for PDX assays) repeating intervals. The number of caspase 3/7 positive cells (apoptotic target cells) per millimeter squared over time was enumerated for all conditions up to 72 h after T-cell addition using IncuCyte ZOOM 2018A software (Sartorius)—dying T cells were gated out by size exclusion.
Plates were imaged from this point using the phase and green fluorescence channels in the IncuCyte ZOOM system (Sartorius) with 10× objective lens at 2-h (3 h for PDX assays) repeating intervals. The number of caspase 3/7 positive cells (apoptotic target cells) per millimeter squared over time was enumerated for all conditions up to 72 h after T-cell addition using IncuCyte ZOOM 2018A software (Sartorius)—dying T cells were gated out by size exclusion.
9
Scratch Wound Assay with Incucyte ZOOM
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An Incucyte® ZOOM System (Sartorius, Göttingen, Germany) was used for the scratch test. WT and TP53-KO cells were seeded in an Incucyte® Imagelock 96-well plate at a density of 2.0 × 106 cells per well and grown until they reached a full monolayer (24 h). To suppress proliferation, the cells were pretreated with mitomycin C. In brief, 2 h prior to wounding, the culture medium was replaced with serum-free medium containing 10 µg/mL mitomycin C (Merck, Rahway, NJ, USA). The injury was inflicted using an IncuCyte 96-pin wound-making tool (Essen Bioscience, Göttingen, Germany). Once the injury was inflicted, cells were rinsed twice with fresh medium and cultured for 24 h. Images were captured hourly. The wound area was calculated using Incucyte® ZOOM Software (v. 2016A) (Sartorius, Germany).
10
Real-time Migration Assay with IncuCyte ZOOM
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A real-time migration assay was conducted using the IncuCyte ZOOM system (Sartorius, Göttingen, Germany) in 96-well plates according to the manufacturer’s instructions. Briefly, cells were seeded in triplicate at a density of 1 × 104 cells/well and scratch wounds were made using the IncuCyte WoundMaker (Sartorius) after cells reached 90% confluence. The plates were monitored every 3 h via live cell imaging and time-lapse images. Results were presented as wound confluence (%) (i.e., percentage of the wound area occupied by cells). The mean value of the 3 replicates in each sample (PSP, n = 3; BHDS, n = 3) was reported as the representative value.
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