Oncology

Cancer cell counting & analysis assays

Whether researchers are working on uncovering either the genetic or environmental factors affecting cancer development, studying individual cancer cells is key in identifying possible causes of individual cancer types. Since cancer is one of the leading causes of mortality worldwide, developing novel treatments to fight cancer is of huge importance. For this reason, cancer research requires fast, reliable, and detailed cell analysis.

The NucleoCounter® NC-3000™ provides a fast cell cycle analysis and a comprehensive array of plug-and-play apoptosis assays allowing the user to carry out detailed analyses of cancer cell vitality. NucleoCounter® instruments can also determine the viability and cell count of cancer cells with extreme precision and speed, even when they are grown in spheroids or on microcarriers.

Analyzing cell cycles

Unlike normal cells, cancer cells undergo uncontrolled cell division due to mutations in genes that would usually regulate the cell cycle, often leading to solid tumors. Therefore, studies into the cell cycle are essential for understanding cancers and they are one of the most powerful tools in this field of research.

The NucleoCounter® NC-3000™ provides a fast and easy cell cycle analysis in less than five minutes called the Two-Step Cell Cycle Assay. After adding a lysis buffer supplemented with fluorescent DNA stain, all cell nuclei will be stained. The NC-3000™ can then measure and analyze the sample.

The instrument’s accompanying NucleoView™ software instantly displays a cell cycle profile, enabling you to identify events in the sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase. With its FlexiCyte™ software package, the NucleoCounter® NC-3000™ can even be used to study cell proliferation. By detecting BrdU and EdU incorporation using fluorescently labeled antibodies, this assay allows for advanced studies of cell proliferation.

The different cell cycle phases can be distinguished based on the DNA content using a fluorescent DNA stain.

The two-step cell cycle assay of untreated and camptothecin (CPT) treated Jurkat cells, shows the 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. CPT is a topoisomerase inhibitor, and some of its derivatives are used as anti-cancer drugs.

Diagram of the cell cycle (left) and diagrams showing the distribution of cells in the cell cycle phases under different conditions (right).

Plug & play assays with the NucleoCounter® NC-3000™

Cell Counting
Cell count and viability assays: High-precision cell count of mammalian, insect and yeast cells from a variety of sample and culture types.

Apoptosis

Mitochondrial Potential Assay: Early apoptosis analysis; detects changes in the mitochondrial potential
Annexin V Assay: Early to mid-apoptosis analysis; detects collapse of phospholipid asymmetry
Caspase Assay: Early to mid-apoptosis analysis; detects changes in caspase 3/7, 8 and 9 activity
Vitality (VB-48) Assay: Late apoptosis analysis; detects changes in reduced thiols
DNA Fragmentation Assay: Late apoptosis analysis; detects fragmented DNA (sub-G1)

Other Assays

Cell Cycle Assay: Unique 5-minute cell cycle assay of live or fixed cells using either DAPI or PI
GFP Transfection Efficiency Assay: Detects GFP-transfected and inviable cells (based on Hoechst and PI)
FlexiCyte™ Assay: User-defined assay setup with optional LEDs, emission filters and settings

Counting cancer cells growing in spheroids

There is a growing need to develop more representative models to perform in vitro testing of cytotoxicity and drug screening in cancer cell biology. This is because conventional 2D cell cultures cannot mimic the complexity and heterogeneity of tumors in vivo.

Several 3D models have been developed, including hanging drops, pellet cultures, spheroid and organoid cultures. These expansion and screening models present a challenge in determining the cell count of cancer cells growing in 3D structures or as organoids containing several cell types to mimic entire cancer tissues.

The NucleoCounter® family of instruments come with cell counting and viability assays specifically made for 3D structures. Using a lysis buffer to break up the 3D structures, the assays ensure a homogenous sample of single nuclei which can then be stained with DAPI for detection and analysis.

Viability & cell count of cancer cells grown on microcarriers

Culturing cancer cells in 3D spheroids can be problematic due to microenvironmental gradients e.g. oxygen and nutrients, limiting cell growth rates. Besides this scaffold-free expansion method of cancer cells in vitro, cancer cells can also be cultivated on the surface of microcarriers and inside of macroporous microcarriers. These methods can help us to study the role of cell shape and cell contact in response to cancer treatments.

Determining the concentration and viability of cells grown in and on microcarriers is extremely challenging and requires the time-consuming process of detaching cells with enzymes. The NucleoCounter® instruments offer a unique protocol to count and determine the viability of cells grown directly on microcarriers. You can achieve this by adding a short lysis step just before counting, without the need for an upstream detachment step.

Step-by-step process of counting cells on microcarriers. Cells are lysed, stained with DAPI, counted, and verified.

Cells are lysed, bringing the nuclei into suspension (Image A). The total number of cells will be stained with DAPI and detected by a NucleoCounter® (Image B). The software analysis enables the user to verify that all cells have been counted correctly (Image C). Afterwards, the non-viable cells are stained with DAPI without any pretreatment with a lysis buffer as all dead cells without cell adhesions are free in suspension and no longer attached to the microcarriers.