Dual-fluorescence Viability(AO/PI), Acridine orange (AO) and propidium iodide (PI) are nuclear nucleic staining and acid-binding dyes. AO can penetrate the membrane of both dead and living cells and stains the nucleus, generating a green fluorescence. In contrast, PI can only permeate the disintegrating membranes of dead nucleated cells, generating red fluorescence. The image-based technology of the Countstar Rigel excludes cell fragments, debris, and artifact particles as well as undersized events such as platelets, giving a highly accurate result. In conclusion, the Countstar Rigel system can be used for every step of the cell manufacturing process.

T/NK Cell-Mediated Cytotoxicity, In the recently FDA-approved CAR-T cell therapy, genetically engineered T-lymphocytes bind specifically to the targeted cancer cells (T) and kill them. The Countstar Rigel analyzers are able to analyze this complete process of T/NK Cell-Mediated Cytotoxicity.

Cytotoxicity studies are performed by labeling the target cancer cells with CFSE or transfecting them with GFP. Hoechst 33342 may be used to stain all cells (both T cells and tumor cells). Alternatively, target tumor cells can be stained with CFSE. Propidium iodide (PI) is used to stain dead cells (both T cells and tumor cells). Discrimination between different cells can be obtained using this staining strategy.

GFP Transfection Efficiency, In molecular genetics, various model organisms, and cell biology, the GFP gene is frequently used as a reporter for expression studies. Currently, scientists are commonly using fluorescent microscopes or flow cytometers to analyze the transfection efficiency of mammalian cells. But handling the complex technology of an advanced flow cytometer demands an experienced and highly qualified operator. Countstar Rigel enables users to easily and accurately perform a transfection efficiency assay without the operation and maintenance costs associated with traditional flow cytometry.

Cell Apoptosis, The progress of cell apoptosis can be monitored using FITC conjugated Annexin-V in combination with 7-ADD. Phosphatidylserine (PS) residues are normally located at the inner side of the plasma membrane of healthy cells. During early apoptosis, the membrane integrity gets lost and PS will be translocated to the outside of the cell membrane. Annexin V has a strong affinity to PS and is therefore the ideal marker for early apoptotic cells.

Cell Cycle, During cell division, cells contain increased amounts of DNA. Labeled by PI, an increase in fluorescence intensity is directly proportional to an accumulation of DNA. The differences in the fluorescence intensities of the single cells are the indicators of the actual status of the cell cycle MCF 7 cells were treated with 4μM of Nocodazole to arrest these cells at different stages of their cell cycle. The bright-field images acquired during this test scenario allow us to identify each single cell. The PI fluorescence channel of the Countstar Rigel identifies the DNA signals of single cells even in aggregates. A detailed analysis of the fluorescence intensities can be carried out using the FCS.

CD Marker Phenotyping, The Countstar Rigel models offer a faster, simpler and more sensitive approach to,immuno-based phenotyping of cells more efficient. With high resolution images and powerful integrated data analysis capabilities, the Countstar Rigel allows users to achieve consistently reliable results without the need for extensive complex control settings and fluorescence compensation adjustments.

The Cytokine Induced Killer (CIK) cell differentiation demonstrates the outstanding performance quality of the Countstar Rigel analyzer in direct comparison to high class flow cytometers. PBMCs of mouse in culture were stained with CD3-FITC, CD4-PE, CD8-PE, and CD56-PE, and induced by Interleukin (IL) 6. Then analyzed simultaneously with Countstar® Rigel and Flow Cytometry. In this test, the CD3-CD4 , the CD3-CD8, and the CD3-CD56 were divided into three groups, to determine the proportion of different cell subpopulations.

Detection of Degenerated Cells by Immunofluorescence, Monoclonal antibodies producing cell lines will lose some positive clones during cell proliferation and passaging due to degradation or genetic mutations. A higher loss will significantly affect the productivity of the manufacturing process. The monitoring of the degradation plays an important role in the process control to shift the yield of antibodies to the optimum.

Most of the antibodies manufactured in the BioPharma industry can be detected by immunofluorescence labeling and analyzed quantitatively by the Countstar Rigel series. The bright-field and fluorescence channel images below clearly show those clones that lost their attribute to produce the desired antibodies. The more detailed analysis with the DeNovo FCS Express Image software confirms, that 86.35 % of all cells are expressing the immunoglobulins, only 3.34 % are clearly negative.

Trypan (capitalize B in Blue) Cell Counting, Trypan blue staining is still used in the majority of cell culture labs.

The Trypan Blue Viability and Cell Density BioApp can be installed on all Countstar Rigel models. Our protected image recognition algorithms analyze more than 20 parameters to classify each single object detected.

Cell Line Storage QC, In cell storage, a sophisticated quality management concept ensures safe, efficient monitoring of all cellular products. This guarantees the stable quality of cell cryopreserved, cryo-preserved for experiments, process development, and production.

The Countstar Rigel acquires high-resolution images, analyzing various morphological characteristics of the cellular objects such as diameter, shape, and aggregation tendency. Images of different process steps can be easily compared to each other. So variations in shape and aggregation can easily be detected, by avoiding subjective human measurements. And the Countstar Rigel database has a sophisticated management system for the storage and retrieval of images and data.