When 50–60% confluent in T25 flasks, both LAPC-4-AI and PC-3 cells were treated with vehicle control, 40μM EGCG+5μM Q, 5nM Doc, or EGCG+Q+Doc for 48h. Cells were trypsinized and monolayers attaching to the bottom were collected. The procedures for cell cycle and apoptosis analysis using a small cytometer Cellometer Vision (Nexcelom Bioscience LLC, Lawrence, MA) were described previously [29 (link), 30 (link)] with minor modifications. Briefly, for cell cycle assay cells were centrifuged and pellet resuspended and fixed in cold methanol. The cells were centrifuged again and pellet was stained in propidium iodide (PI) solution (Nexcelom Bioscience LLC) for imaging cytometry using Cellometer Vision. For apoptosis assay, cells were centrifuged and pellets were resuspended in Annexin V binding buffer and double-stained with Annexin V-FITC (Nexcelom Bioscience LLC) and PI (Nexcelom Bioscience LLC) for Cellometer analysis. A positive control was generated by heating cells in a 45°C water bath for 10min. Non-treated cells were used as negative control. Both controls were processed with the samples. The fluorescence data generated by the Cellometer software were converted into FCS files and analyzed by De Novo FCS Express 4 software (Los Angeles, CA). The experiment was performed in triplicate.
Cellometer vision
Manufactured by Revvity
Sourced in United States
The Cellometer Vision is a compact, automated cell counter that utilizes image cytometry technology to accurately measure cell concentration and viability. It provides rapid and reliable cell counting results for a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Annexinv fitc dye, by BioLegend (2 mentions)
Pi dye, by BioLegend (2 mentions)
Fcs express 4, by De Novo Software (2 mentions)
Viastain aopi reagent, by Revvity (2 mentions)
Trypan blue, by Corning (1 mentions)
Annexin 5 alexa fluor 488 pi apoptosisassay kit, by Thermo Fisher Scientific (1 mentions)
Cytokine mouse magnetic 10 plex kit, by Thermo Fisher Scientific (1 mentions)
Accumax, by Innovative Cell Technologies (1 mentions)
Pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
3h leucine, by PerkinElmer (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Staurosporine, by Merck Group (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Methanol, by Merck Group (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Bz 9000, by Keyence (1 mentions)
Annexin 5 fitc, by Revvity (1 mentions)
Cellometer sd100, by Revvity (1 mentions)
37 protocols using cellometer vision
1
Cell Cycle and Apoptosis Analysis of Prostate Cancer Cells
2
Cell Growth Kinetics and Morphometry
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The P6 cell output was subcultured as per the 3K3D and 4K3D schemes in T25 flasks and counted on each day until confluence. Lag period, doubling time and saturation density were estimated by the standard growth curve method. Similar estimations were made for the cells of P5 and the clone after plating them at a density of 3000 cells per cm2. The saturation density of 3K3D and 4K3D cells was additionally estimated in T75 flasks.
The 3rd day cells were analyzed for cell size using Cellometer Vision (Nexcelom Bioscience LLC, Lawrence, MA, USA) which was previously calibrated with beads of known sizes. In brief, the trypsinized cells were pipetted into the disposable counting chambers, bright-field images were automatically captured and the pixel area of the captured cells was converted to corresponding cell diameter.
The 3rd day cells were analyzed for cell size using Cellometer Vision (Nexcelom Bioscience LLC, Lawrence, MA, USA) which was previously calibrated with beads of known sizes. In brief, the trypsinized cells were pipetted into the disposable counting chambers, bright-field images were automatically captured and the pixel area of the captured cells was converted to corresponding cell diameter.
3
Multicultivator Cell Culture Characterization
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Cell cultures were grown in MC-1000 multicultivators in BG11 and YBG11-Fe under 200 or 800 μmol photons m -2 s -1 as mentioned above. The cells were harvested after three days, diluted to OD730 = 0.01 and the cell number was counted on an automated cell counter (Cellometer Vision; Nexcelom) with further manual curation to improve accuracy.
4
Measuring Cell Growth Rates
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To measure the cell growth rates, we diluted cells from log-phase growth (1–1.5 × 106 cells/ml) into the fresh growth media at 0.1 × 106 cells/ml and incubated at 22 °C with constant shaking at ~ 180 rpm. Cell growth was monitored by counting cells using a cell counter machine [Cellometer Vision-Nexcelom Bioscience] at regular time intervals over several days. Cell growth assays were performed in triplicate sets in each independent experiment (N = 3).
5
Cell Viability Assay for Melanoma Cells
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PCS-200-013, MEL-F-NEO, SK-MEL-2 and MeWo cells (4×104) were cultured in T25 flasks and treated with either an equal volume of sterile water (vehicle control) or ACPD or DNDA (0.1–3.5 µM). Additional doses of sterile water or ACPD or DNDA were supplied every 24 h during a 3-day incubation period and cells were subsequently lifted using Trypsin-EDTA solution (1.5 ml/flask) and neutralized with the equal volume of media. Subsequently, live cells were counted using the Scepter, an automated cell counter from Millipore (Billerica, MA, USA) at 24-h intervals. Cell counts obtained from Scepter were compared with the counts obtained from the Cellometer Vision from Nexcelom Bioscience (Lawrence, MA, USA).
6
Rapid Cell Viability Quantification
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The cell viability was determined by the AO/PI staining assay (Nexcelom Bioscience, cat# CS2‐0106‐5mL, Lawrence, MA, USA) following the manufacturer's suggested protocol. Briefly, total cells were harvested by trypsinization and centrifugation and resuspended with DMEM medium. For AO/PI staining, took 25 μL of AO/PI staining solution to a 1.5 mL Eppendorf tube; added 25 μL of the cell suspension to the same tube and mixed by pipetting up and down; immediately loaded 20 μL mixture into a disposable counting chamber (SD100 Slides, Nexcelom Biosciences, Lawrence, MA, USA) and analyzed with the cell counter (Cellometer Vision, Nexcelom Bioscience).
7
Trypan Blue Cell Viability Assay
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The reaction mixture for Trypan blue assay was prepared by mixing 1 part of 0.4% Trypan blue (25-900-CI, Corning) and 1 part of diluted cell samples. The mixture was incubated for 1 minute, and cells were counted on Cellometer Vision (Nexcelom, Vision-310-0208).
8
Apoptosis and Necrosis Quantification
9
Lymphocyte Cytokine Profiling Assay
10
Plasma Membrane Resealing Kinetics
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Permeability to propidium iodide (PI) was used to measure the kinetics of plasma membrane resealing after nanoelectroporation. Immediately after nsEP exposure all samples were diluted 1:1 with RPMI and placed at 37 °C in the water bath or on ice. At 0, 10, or 30 min post exposure, 20 μl of each cell sample was mixed with an equal volume of 50 μg/ml PI (Sigma) in PBS and placed at 37 °C for 5 min. Cell samples were loaded into a counting chamber of Cellometer Vision (Nexcelom Bioscience LLC, Lawrence, MA) and both bright field transillumination and fluorescence images were acquired. The cell diameters and PI fluorescence intensity of 300–500 cells per sample were measured from the image and logged using Cellometer software. Images were generated using Grapher 11 (Golden Software, Golden, CO).
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