Application Note

Evaluating cell cycle inhibitors using a live cell assay

  • Maintain live cells inside instrument for up to 72 hours
  • Minimize hands-on time
  • Segment cells using brightfield images
  • Classify cells transfected with a cell cycle sensor
  • 活细胞在仪器内持续生长观察 长达72小时以上
  • 减少实验操作时间
  • 明场无标记下识别细胞
  • 应用细胞周期转染试剂检测周期变化

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Introduction

Monitoring treatment effects on the cell cycle is particularly relevant to progressing oncology research and drug discovery. For instance, compounds known to inhibit mitosis are often utilized to slow division of cancer cells. High-content screening assays using live cells have been developed to enable the classification of cells by their cell cycle phase. The technology uses the BacMam delivery system to transiently transfect cells with two fluorescent fusion proteins that are regulated by cell cycle.

This time-lapse assay was run for 2-3 days inside an ImageXpress® Micro High-Throughput Imaging system equipped with environmental control, which maintained the living cells for the entire duration of the experiment. At specified time intervals, both brightfield and fluorescent images of live cells were collected. The complete workflow includes an automated analysis of the time-lapse images with an integrated software analysis module that identifies all cells based on the brightfield image and then classifies each cell based on the expression of fluorescent proteins that are present at different phases of the cell cycle.

Visualize fluorescent proteins indicating phase of cell cycle

Cell cycle was monitored over time by using the reagent Premo™ FUCCI Cell Cycle Sensor (Thermo Fisher). Immediately before plating, the cells were transfected with fluorescently-labeled cell cycle-regulated proteins, geminin and Cdt1. Geminin-emerald GFP and Cdt1-tagRFP emit at the wavelengths corresponding to FITC and TRITC, respectively. Since Cdt1 and geminin are only present during specific phases of the cell cycle, their expression in the nuclei can be used to classify cells by cell cycle phase (Figure 1).

Figure 1. Fluorescent cellular changes associated with FUCCI Cell Cycle Sensor. Cells in G1 express Cdt1-RFP (red). Cells in G2/M express Geminin-GFP (green). Cells in S phase appear yellow (both Geminin-GFP and Cdt1-RFP expressed).

Method

  1. To prepare cell cultures, a suspension of HeLa cells at 40,000 cells/mL was pre-mixed with 30 particles/cell of each FUCCI reagent and plated into a 96- well plate at a density of 4,000 cells/ well. The plate was incubated for ~8 h at 37°C, 5 % CO2 to allow attachment.
  2. The cells were treated with various concentrations of cell division inhibitors. Then the plate was loaded into the ImageXpress Micro system.
  3. Images were acquired on the ImageXpress Micro sytem at 2-3 h intervals with a 20x Plan Apo objective. At each time interval, the instrument was configured to acquire an image in brightfield and fluorescent wavelength channels, FITC and TRITC. Image acquisition was stopped after 48-72 h to allow cells to complete 1-2 divisions.
  4. Time-lapse images were analyzed within the Custom Module Editor of MetaXpress® High-Content Image Acquisition and Analysis Software.

Identify cells with brightfield images and classify cells using fluorescent cell cycle sensor

Using the FUCCI cell cycle sensor in living cells, changes in cell cycle phase can be measured over the duration of the compound treatment, providing more information than a fixed endpoint analysis. Time-lapse images of HeLa cells transfected with the FUCCI cell cycle sensor were acquired and analyzed using a MetaXpress analysis module that identifies cells in the brightfield image and then classifies each cell based on the expression of fluorescence in their nuclei. Cells in G1 phase express red fluorescence, cells in G2/M phase express green fluorescence, and cells in S phase express both red and green fluorescence (Figure 2). Cells in G0 do not emit fluorescent signals, but are still detected in the brightfield image and included in the analysis for total cell counts. In addition, individual cells can be tracked through each cell cycle phase over time if desired.

Figure 2. Time-lapse images of HeLa cells transfected with the FUCCI cell cycle sensor. The images were analyzed using a MetaXpress software module that identifies cells in the brightfield image and then scores them based on the expression of fluorescent cell cycle indicators in the nuclei: G1 = red only, G2/M = green only, S = red + green, G0 = non-fluorescent. Representative overlay images from time points 2 and 6 are shown for cells dosed with 2 nM Paclitaxel, 10 nM Nocodazole, and 100 nM Colchicine. The segmentation masks illustrate which cells were identified in each phase of the cell cycle

Cell cycle inhibitors cause cell arrest in specific phases of the cell cycle.

Analysis of each time point was accelerated using MetaXpress PowerCore parallel processing capabilities. After 24 hours of compound treatment, the majority of cells in the control wells were resting in G0 and only a minimal number of cells expressed cell cycle markers. Cells treated with Paclitaxel and Nocodazole demonstrated a significant number of cells in G2/M phases, where they remained in cell arrest for the duration of the timecourse (Figure 3)

Figure 3. Effect of compound treatment on cell cycle phase over duration of 48 h. Paclitaxel treatment (2 nM) shows the percentage of cells in S, G2, and M is significantly higher than the control. Treatment with 10 nM Colchicine shows percentage of cells in G2 and M is significantly higher than the control. Treatment with 100 nM Nocodazole shows a significant inhibition from dividing (also seen as no increase in cell number over the course of the experiment – data not shown) and half the cells remained arrested in G2/M Phases.

Summary

The Premo™ FUCCI Cell Cycle Sensor reagent used in conjunction with the ImageXpress Micro system equipped with environmental control and MetaXpress software enables an accurate measurement of cell cycle phase in living cells with a highly efficient workflow. The high-throughput screening technology offers scientists a fast and robust image-based quantitation of the cell cycle markers, while maintaining cell integrity for long time course durations. Furthermore, the use of the MetaXpress software provides a capability to analyze brightfield images, eliminating the use of toxic cellular stains for live cells. Consistent and statistically relevant quantitative analysis allows for the testing of multiple compounds at differing concentrations and can be suited to the parameters of any cell cycle assay

前言

监测对细胞周期变化的影响对于肿瘤的发展及药物研发有着重要的作用。例如，已知抑制有丝分裂的化合物大都用来减缓肿瘤细胞的生长。活细胞高内涵的筛选已被验证可以区分细胞的每个周期。此技术结合BacMam转染系统，可使细胞表达两种细胞周期相关荧光融合蛋白。

延时监测实验持续2-3天，细胞置于 ImageXpress® Micro高内涵成像与分析系 统的环境控制舱室中，整个实验都可维持 活细胞的正常生长环境。按预先设置的时 间间隔，同时获取明场和荧光的活细胞图 像。这个完整的实验方案包括了预置模块 对延时拍摄的图片进行分析，基于明场的 图像识别所有细胞，基于荧光蛋白的表达 区分不同的细胞周期。

可视化荧光蛋白表明细胞所处周期

使用实时监测细胞周期Premo™ FUCCI细 胞周期指示剂(Thermo Fisher)。在种板之 前，细胞转染荧光标记的细胞周期相关蛋 白，geminin和Cdt1。Geminin为绿色的 GFP，Cdt1为红色RFP，拍摄通道分别为 FITC和TRITC。由于geminin和Cdt1都只 在特定的周期表达，因此，他们在细胞核 中的表达可以指示细胞所处的细胞周期(图 1)。

图1.FUCCI细胞周期标记物标记后与细胞周期变化关系。 表达Cdt1-RFP红色为G1期，表达Geminin-GFP绿色为G2/M期，同时表达Cdt1-RFP红色和Geminin-GFP绿色的为S期。

方法

  1. 准备细胞悬液，40,000细胞/mL的Hela 细胞悬液与30颗粒/细胞浓度的FUCCI试 剂混合，以4,000细胞/孔的浓度种在96 孔板中，孔板置于37°C，5 % CO2的环境 下贴壁8小时。
  2. 不同浓度的有丝分裂抑制剂处理细胞， 然后孔板放入ImageXpress Micro系统。
  3. . 每隔2-3小时系统自动拍摄一次图片， 使用20倍Plan Apo物镜，同时获取明场 及两个荧光通道FITC和TRITC的图像。 实验持续48-72小时，细胞可完成1-2次 分裂。
  4. 分析延迟实验图像使用MetaXpress®高内涵成像与分析软件的用户自定义模块。

明场识别细胞，荧光蛋白区分细胞周期

活细胞使用FUCCI细胞周期指示剂，化合物处理后细胞周期的变化可以被实时监测到，比终点法检测固定细胞能得到更多的信息。转染了FUCCI细胞周期指示剂的Hela细胞在高内涵细胞中实时拍摄了图像，并通过MetaXpress®高内涵成像与分析软件的模块进行分析，明场下识别出所有细胞，基于荧光蛋白表达区分出不同周期。表达红色为G1期，表达绿色为G2/M期，同时表达红色和绿色的为S期(图2)。处于G0期的细胞没有荧光信号，但是会在明场下被识别到并计入细胞总数中。另外，分裂的细胞也可被记录。

图2.Hela细胞转染FUCCI细胞周期指示剂后的延时成像。 这些图片并通过MetaXpress®高内涵成像 与分析软件的模块进行分析，明场下识别出所有细胞，基于荧光蛋白表达区分出不同周期。表达红 色为G1期，表达绿色为G2/M期，同时表达红色和绿色的为S期，G0期为无荧光。图中所示为第二和 第六个时间点的图片，细胞被2 nM Paclitaxel, 10 nM Nocodazole, and 100 nM Colchicine处理。分 割的蒙版图显示了被识别到的不同时期的细胞。

细胞周期抑制剂可引起细胞停滞在特定周期

分析过程可使用MetaXpress PowerCore 并行处理软件加速分析速度。经过24小时 化合物处理，大部分细胞停留在G0期，只 有少数细胞表达了细胞周期标记蛋白。被 紫杉醇和诺考达唑的细胞有一定的细胞停 滞在G2/M期(图3)。

图3. 经过48小时化合物处理对细胞周期的影响。 Paclitaxe处理组（2nM）处于S、G2和S期的细胞明 显高于对照组，10 nM Colchicine处理组处于G2和M期的细胞明显高于对照组，100 nM Nocodazole 有抑制分裂的作用(并且在实验过程中细胞数量没有增长)，且半数细胞停滞在G2/M期。

总结

Premo™ FUCCI细胞周期指示剂结合配置 环境控制的ImageXpress Micro成像系统 和MetaXpress软件，可实现高效的活细胞 周期精确测量。高通量筛选技术可为科学 家提供一个快速的、自动的基于图像定量 分析细胞周期的方法，并能够完整的记录 长时间内的细胞周期变化。另外， MetaXpress软件能够在明场图像识别细 胞，避免了染料对活细胞的毒性。同一标 准具有统计学意义的定量分析方法可评价 多化合物在不同浓度对细胞周期影响的相 关参数。

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