Determination of Cell Count and Viability of Cancer Cells Grown on Microcarriers using NucleoCounter® Instruments

Introduction

Cancer is regarded as one of the major causes of mortality across the world and therefore it is important to develop novel treatments for this deadly disease. Studying cancer-affected cells and healthy cells is one of the key approaches to determine the potential causes of cancer.

Rapid, reliable, and in-depth analysis is essential for cancer research. The NucleoCounter® instruments offer solution platforms from precise cell count to viability determination and advanced cell analysis.

With the NucleoCounter® instruments, cell count and viability of cancer cells can be precisely determined even when growing on microcarriers or in spheroids. Fast cell cycle analysis, as well as a wide range of plug-and-play apoptosis assays, can be used for in-depth analysis of cancer cell vitality.

Cell cycle analysis of cancer cells

When compared to healthy cells, cancer-affected cells undergo uncontrolled cell division owing to mutations in genes that regulate the cell cycle, and this causes solid tumors.

Cell cycle studies are, therefore, essential for understanding cancer and are widely regarded as one of the most powerful tools in the cancer research field. Using the NucleoCounter® NC-250™ and NucleoCounter® NC-3000™, cell cycle analysis can be easily and rapidly performed within five minutes (Figure 1).

Once a lysis buffer is added, the next step is to stain all cell nuclei and measure the sample using the NucleoCounter® instruments. The integrated NucleoView™ software will readily display a cell cycle profile, which allows identification of events in the sub-G1-phase, G0/G1-phase, S-phase, and G2/M-phase.

Thanks to the FlexiCyte™ software package, even cell proliferation can be studied using the NucleoCounter® NC-3000™, for example, BrdU und EdU incorporation determined with fluorescently labeled antibodies. This allows for advanced cell proliferation studies.

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.

Determination of apoptosis and necrosis in cancer cells

Destroying cancer-affected cells without causing damage to healthy cells is the ultimate objective of cancer research. And so, it is essential to understand the detailed cell death mechanism in both healthy and cancer cells.

With the NucleoCounter® NC-3000™, users can easily determine whether cells are early- or late-apoptotic, whether cell death is due to apoptosis or necrosis, and whether it is an extrinsic or intrinsic pathway of apoptosis.

The mechanism of cell death can be comprehensively studied using an array of 5 plug-and-play assays, including mitochondrial potential with JC-1, Annexin V, DNA fragmentation, caspase signaling, and the one-minute vitality assay, which covers early to late apoptosis.

Counting of cancer cells growing in spheroids

There is a growing need to create more representative models in order to conduct in vitro analysis of cytotoxicity and drug screening in the field of cancer cell biology. This is because of the incapability of conventional 2D cell cultures to mimic the heterogeneity and complexity of tumors in vivo.

There are several 3D models available, including pellet cultures and hanging drops. Such screening and expansion models pose a challenge to determine the count of cancerous cells growing in spheroids.

The NucleoCounter® line of instruments such as NC-200™, NC-250™, and NC-3000™ are equipped with an assay particularly designed for spheroids (Figure 2). The “Count of Aggregated Cells – A100 and B Assay” is used to break-up the spheroids to obtain a homogenous sample of single nuclei. NucleoCounter® instruments are then used to detect the homogenous sample after staining it with DAPI.

Figure 2. Counting of cancer cells in spheroids. (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.

Cell count and viability of cancer cells grown on microcarriers

Cancer cell culturing in 3D spheroids presents a problem of microenvironmental gradients, for example, nutrients and oxygen limiting the rate of cell growth.

In addition to this scaffold-free expansion method of cancer cells in vitro, it is possible to cultivate cancer cells inside of macroporous microcarriers and on microcarriers to investigate the role of cell contact and cell shape in response to cancer treatments.

It is a challenging task to determine the viability and concentration of cells grown on and in microcarriers, requiring time-intensive detachment of cells using enzymes.

The unique protocol provided by the NucleoCounter® instruments NC-200™, NC-250™, and NC-3000™ does not require previous detachment to determine the viability and count of cells grown on microcarriers (Figure 3).

Figure 3. Viability and cell count of cancer cells on microcarriers. (A) The addition of Reagent A100 and B lyses the cells bringing the nuclei into suspension. The total number of cells will be stained with DAPI and can be detected by the NucleoCounter® family instruments NC-200™, NC-250™ and NC-3000™. Afterwards, the dead count will be determined. (B) Image cytometry of cells stained with DAPI shows only the nucleated cells. (C) The accompanying NucleoView™ software allows the user to verify that all cells have been counted correctly.

The cell nuclei are brought into suspension by lysing the cells with the “Viability and Cell Count – A100 and B Assay.” The next step is to stain the total number of cells with DAPI and detect them using the NucleoCounter® instruments.

This is followed by staining the non-viable cells with DAPI with no pretreatment as it is assumed that without cell adhesions, all dead cells are freely suspended and no longer connected to the microcarriers.

About ChemoMetec A/SChemoMetec A/S

Founded in 1997, Chemometec specialises in the design, development and production of high quality instruments using patented technology for a wide range of applications in cell counting and evaluation.

Chemometec work closely with life science companies, research institutes, universities, hospitals, specialist clinics and a wide range of food and beverage manufacturers, matching our technical development expertise to customer needs - with quality results, reliability, cost-efficiency and ease-of-operation as our guiding principles.

Chemometec support a growing customer base worldwide with responsive technical services and the ready availability of a wide range of consumables.


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Last updated: Mar 1, 2017 at 10:07 AM

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