Live-cell imaging is an extremely efficient technique that allows the collection of cellular timelapse information in a single image acquisition. The technique requires the preparation of devices that maintain an optimum environment (temperature, CO2 concentration, humidity control) for preserving cells in their normal condition. This technical note shows the evaluation results of the internal stage incubator of the CellVoyager CV8000 high-content analysis system. The evaluation consisted of the investigation of cell proliferation variation within well plates, and comparison against a conventional CO2 incubator.
Fig 1(a). Timelapse movie: A1 well (72hrs)
Fig 1(b). Timelapse movie: B2 well (72hrs)
Fig 1(c). Timelapse movie: C3 well (72hrs)
Fig 1(d). Timelapse movie: D6 well (72hrs)
- HeLa cells stably expressing Azami-Green (green fluorescent protein) were seeded (500 cells/well) in 250uL of medium in 96-well plates (Greiner #655896), and incubated for 24hrs. (Two plates were prepared.)
- One plate was incubated for 72 hours in a conventional CO2 incubator. Imaging was performed at 0hrs and 72hrs using the CV8000.
The other plate underwent live cell imaging using the CV8000. Whole well imaging was performed at 3hr intervals for 72hrs with a 4x objective lens.
- Analysis was performed using the high-content analysis software CellPathfinder.
The total area covered with cells in each well was calculated to evaluate the proliferation of cells. The variation of proliferation rate across wells was investigated. A comparison with the conventional CO2 incubator was also made.
Fig 1(e). Line graphs of cell proliferation for each well
Vertical axis: total area (total area of cells in that well) Horizontal axis: time (0-72hrs)
Cell proliferation deteriorated in the plate’s corner wells; however, it continued at a good rate in the other wells.
Time-lapse movie under the similar condition: comparison of each wells: Play
Fig 2. Variation of cell proliferation rates within the well plate (n=3)
(A) Total area ratio for each well (total area at 72hrs / total area at 0hrs)
96 well average: 7.7
96 well coefficient of variation (standard deviation/average): 8.5×10-2
36 well coefficient of variation (perimeter wells): 1.1×10-1
60 well coefficient of variation (except perimeter wells): 5.1×10-2
(B) Change in total area ratio over time (error bars show standard deviation)
After 24hrs, there was no variation in the total area ratio across groups. Even after 72hrs, perimeter wells (except corner wells) showed similar results to the center wells.
Fig 3. Proliferation ratio comparison with a conventional CO2 incubator (n=3)
Comparison of the cell proliferation ratio 72hrs after seeding, for each well, between a regular CO2 incubator and the CV8000
CV8000 total area ratio / CO2 incubator total area ratio×100
(The nearer the value to 100, the more similar cell proliferation was between the CV8000 and the CO2 incubator.)
96 well average: 90
36 perimeter well average: 81
60 well average (except perimeter wells): 96
The results showed there was little variation in cell proliferation across wells (except perimeter wells) in the CV8000’s internal incubator, and its performance was comparable to that of a conventional CO2 incubator. It is generally understood that cell proliferation rates deteriorate on the perimeter of well plates due to culture medium evaporation. In this evaluation, however, a large variation in proliferation rates between the perimeter wells (except corner wells) and center wells was not observed. These results indicate that the CV8000’s internal incubator possesses sufficient performance for live-cell imaging.
CellPathfinder 是專為Yokogawa HCA 系統、CQ1 和 CellVoyager 系列而設計的軟體。 從初學者到專家，都可讓您量化細微的生理變化，使用各種圖形選項量化無標籤樣本。
CV8000 High-Throughput System 高內涵影像分析系統
CellVoyager CV8000 是一款高階的高內涵分析系統，使用橫河特有的高速共軛焦掃描儀。具備水鏡、多達四個高視野相機、細胞培養環境的載物台和自動化分液器的組合，不僅實現高內涵、高解析，也可以使用更複雜的評估系統進行表型篩選。
High Content Analysis CellVoyager
我們的高內涵分析 (HCA) 系統使用功能強大的軟體，支援從基礎科學到復雜化合物篩選的廣泛研究應用。