がんは世界中で主要な死因の1つとなっており(WHO World Cancer Report 2014)、新たながん治療法の開発は極めて重要です。がん研究は、疫学、分子生物学からがん免疫療法、そして様々ながん治療の手法を評価・比較するための臨床試験の実施まで多岐にわたります。がんの発生に影響を与えているのが遺伝的要因か環境的要因かの特定において、細胞に及ぼしている影響の研究は、がんの原因を特定する主要な方法の1つです。見込みのある治療法が細胞に及ぼす影響を調べるには、迅速で信頼性が高い詳細な細胞解析を必要です。NucleoCounter® -自動セルカウンターは、正確な細胞数と生存率の測定から、高度な細胞解析までのソリューションプラットフォームを提供します。
Figure 1. Fast cell cycle analysis of cancer cells.
- (A) The different cell cycle phases can be distinguished based on the DNA content using a fluorescent DNA stain.
- (B) Two-step cell cycle assay of untreated and camptothecin-treated (CPT) Jurkat cells. The histograms display intensity of the DNA-stain DAPI and can be used to define cell cycle events in the sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase. After CPT treatment, the cell cycle is arrested in the G2/M-phase.
In comparison to normal cells, cancer cells undergo uncontrolled cell division due to mutations in genes regulating the cell cycle leading to solid tumors. Therefore, cell cycle studies are fundamental for understanding cancer and are one of the most powerful tools in cancer research. The NucleoCounter® NC-250™ and NucleoCounter® NC-3000™ provide fast and easy cell cycle analysis in less than 5 minutes (Figure 1). After addition of a lysis buffer, all cell nuclei are stained and the sample can be measured using the NucleoCounter® instruments. A cell cycle profile will readily be displayed in the accompanying NucleoView™ software and events in the sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase can be identified. With the FlexiCyte™ software package, the NucleoCounter® NC-3000™ can even be used for studying cell proliferation with e.g. BrdU und EdU incorporation detected with fluorescently labeled antibodies allowing for advanced studies in cell proliferation.
The essential aim of cancer research is to fight cancer cells without damaging healthy cells. It is therefore of fundamental importance to study the detailed mechanism of cell death in both cancer and healthy cells. Determining whether cell death is caused by necrosis or apoptosis, whether cells are early- or late-apoptotic and whether it is the intrinsic or extrinsic pathway of apoptosis is easy using the NucleoCounter® NC-3000™. A series of 5 plug-and-play assays allow for full investigation of the cell death mechanism including Annexin V, mitochondrial potential with JC-1, caspase signaling, DNA fragmentation and the unique 1-minute vitality assay covering early to late apoptosis (Table 1).
|Plug-and-play assays in the NucleoCounter® NC-3000™|
|Viability and Cell Count Assay||High-precision cell count of cells brought into suspension|
|Viability and cell count - Aggregated cells assay||Viability and cell count of aggregated cells and adipose-derived MSC|
|Viability and cell count, A100 and B||Viability and cell count of heavily aggregated cells and cells growing on microcarriers|
|Viability and cell count - Blood assay||Cell counting in samples containing red blood cells|
|Mitochondrial Potential Assay||Early apoptosis - detects changes in the mitochondrial potential|
|Annexin V Assay||Early to mid apoptosis - detects collapse of phospholipid asymmetry|
|Caspase Assay||Early to mid apoptosis – detects changes in caspase 3/7, 8 and 9 activity|
|Vitality (VB-48) Assay||Late apoptosis – detects changes in reduced thiols|
|DNA Fragmentation Assay||Late apoptosis – detects fragmented DNA (sub-G1)|
|GFP Transfection Assay - Hoechst and PI||Assay to detect GFP-transfected and/or inviable cells in one run|
|2-step Cell Cycle Assays||Unique 5-minute cell cycle assay|
|Fixed cell cycle assay||Traditional cell cycle assay on fixed cells with either DAPI or PI|
|FlexiCyte™||User-defined LEDs, emission filters and settings|
Table 1. Determination of apoptosis and necrosis in cancer cells. Different plug-and-play assays allow for detailed cell analysis in the NucleoCounter® NC-3000™.
- (A) Cells growing in spheroids will be heavily aggregated. By the use of Reagent A100 and B, spheroids will be disaggregated and the membranes permeabilized allowing for staining of nuclei with DAPI.
- (B) Image cytometry of cells stained with DAPI shows the total cell count.
- (C) The accompanying NucleoView™ software allows the user to verify that all cells have been counted correctly.
Growing need arises to develop more representative models to perform in vitro testing of cytotoxicity and drug screening in cancer cell biology as conventional two-dimensional (2D) cell cultures are not capable of mimicking the complexity and heterogeneity of tumors in vivo. Several three-dimensional (3D) models have been developed, including hanging drops and pellet cultures. These expansion and screening models present a challenge in cell count determination of cancer cells growing in spheroids. The NucleoCounter® family instruments NC-200™, NC-250™ and NC-3000™ come with an assay specifically made for spheroids (Figure 2). The use of the “Count of Aggregated Cells – A100 and B Assay” facilitates the break-up of the spheroids, ensuring a homogenous sample of single nuclei which can be stained with DAPI and detected by the NucleoCounter® instruments.
図 3. マイクロキャリア等で増殖する細胞数と生存率の測定。
- (A) Reagent A100およびBを加えることで細胞が溶解し、核が懸濁液に遊離します。
- (B) 全細胞数はDAPIによる青色染色によって、NC-200™、NC-250™またはNC-3000™で検出できます
- (C) 付属のNucleoView™ソフトウェアにより、ユーザーはすべての細胞が正しくカウントされたことを確認できます
Culturing cancer cells in three-dimensional (3D) spheroids comes with the problem of microenvironmental gradients e.g. oxygen and nutrients limiting cell growth rates. Beside this scaffold-free expansion method of cancer cells in vitro, cancer cells can also be cultivated on microcarriers and inside of macroporous microcarriers to study the role of cell shape and cell contact on response to cancer treatments. Determination of concentration and viability of cells grown in and on microcarriers is extremely challenging and requires time-consuming detachment of cells with enzymes. The NucleoCounter® family instruments NC-200™, NC-250™ and NC-3000™ offer a unique protocol to count and determine viability of cells grown on microcarriers without the need of previous detachment (Figure 3). With the “Viability and Cell Count – A100 and B Assay” cells are lysed, bringing the nuclei into suspension. The total number of cells will be stained with DAPI and detected by the NucleoCounter® instruments. Afterwards, the non-viable cells are stained with DAPI without any pretreatment based on the assumption that all dead cells without cell adhesions are free in suspension and no longer attached to the microcarriers.