FlexiCyte™ Assay
Fully Flexible Data Visualization and Analysis
- Advanced cell analysis of a broad range of fluorescent biomarkers and proteins
- Compliant: 21 CFR Part 11/GMP-ready
- Adjustable analysis settings before and after data acquisition
- Integrated image analysis algorithm
- Data visualization and validation during acquisition
Overview of the FlexiCyte™ Assay
The FlexiCyte™ Assay for the NucleoCounter® NC-3000™ advanced image analyzer, enables users to perform detailed cell analysis of a broad range of biomarkers and fluorescent proteins in mammalian cells. The ‘Protocol Adaptation Wizard’ guides the user through a selection of optimal settings.
FlexiCyte™ allows you to build a custom assay using a combination of LEDs from UV to far-red matched with carefully selected emission filters contained within the NucleoCounter® NC-3000™. This allows for simultaneous detection of numerous fluorescent antibodies and proteins.
The ‘Protocol Adaptation Wizard’ guides you through the selection of optimal settings. After image acquisition, data is presented as scatter plots and histograms in the Plot Manager, along with the fluorescent image. Detailed data analysis can then be performed linking individual events between images and plots for full understanding of co-localization of fluorescent events and cell (sub-)populations.
Assay Principle
Samples are stained with fluorophores specified by the user. Note that the default FlexiCyte™ protocol requires counterstaining of the sample with either DAPI or Hoechst-33342 in addition to the user specified staining. DAPI and Hoechst-33342 stain the nucleus and are used for detection (i.e. masking) of the total cell population. Hoechst-33342 stains both living and dead cells and is used for labeling samples with non-fixed cells. DAPI exclusively stains dead cells and is used for labeling cells fixed with either alcohols or aldehydes.
For staining of cellular markers, we recommend using Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 647. These dyes are bright and photostable and have minimal spectral overlap. Avoid using dyes that are prone to photobleaching, such as FITC, PE, APC and PerCP.
For full information and recommendations, please refer to the Application Note: FlexiCyte™ Protocol for the NucleoCounter® NC-3000™ system .
Results Presented in Plot Manager
Use the Plot Manager to analyze the events acquired by the NucleoView™ software with the NucleoCounter® NC-3000™. Draw polygons, quadrants, and markers in the plots to count and gate sub-populations for detailed data analysis and verification of staining events.
Tech notes
Publications
Methyl gallate, gallic acid-derived compound, inhibit cell proliferation through increasing ROS production and apoptosis in hepatocellular carcinoma cells
-
Huang CY, Chang YJ, Wei PL, et al. ,
-
PLOS One ,
-
2021,
-
16(3):e0248521
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Cell Count & Viability Assay, FlexiCyte™ Assay
- Research area: Oncology
Hepatocellular carcinoma (HCC) is a global health problem. Currently, there is no effective therapeutic strategy for HCC. Methyl gallate (MG), from plant-derived phenolic gallic acid, has exhibited antitumor efficacy. However, the effect of MG on HCC is unclear. In vitro...
View PublicationROS Overproduction Sensitises Myeloma Cells to Bortezomib-Induced Apoptosis and Alleviates Tumour Microenvironment-Mediated Cell Resistance
-
Caillot M, Zylbersztejn F, Maitre E, et al. ,
-
Cells ,
-
2020,
-
9(11):2357
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Cell Count & Viability Assay, Cell Cycle Assay, FlexiCyte™ Assay
- Research area: Oncology
Multiple myeloma (MM) is a plasma cell neoplasm that remains incurable due to innate or acquired resistance. Although MM cells produce high intracellular levels of reactive oxygen species (ROS), we hypothesised that they could remain sensitive to ROS unbalance. We...
View PublicationBisphenol A Diglycidyl Ether (BADGE) and Bisphenol Analogs, but Not Bisphenol A (BPA), Activate the CatSper Ca2+ Channel in Human Sperm
-
Rehfeld A, Andersson AM, Skakkebæk NE ,
-
Frontiers in Endocrinology ,
-
2020,
-
11:324
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Sperm Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Aim: Evidence suggests that bisphenol A diglycidyl ether (BADGE), bisphenol A (BPA), and BPA analogs can interfere with human male fertility. However, the effect directly on human sperm function is not known. The CatSper Ca2+ channel in human sperm controls...
View PublicationThe calcium-sensing receptor is essential for calcium and bicarbonate sensitivity in human spermatozoa
-
Boisen IM, Rehfeld A, Mos I, et al. ,
-
The Journal of Clinical Endocrinology and Metabolism ,
-
2020,
-
dgaa937
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Sperm Cells
- Assay: FlexiCyte™ Assay
- Research area: Animal Reproduction
Context: The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in...
View PublicationPropyl Gallate Inhibits Hepatocellular Carcinoma Cell Growth Through the Induction of ROS and the Activation of Autophagy
-
Po-Li Wei , Chien-Yu Huang , Yu-Jia Chang ,
-
PLoS One ,
-
2019,
-
14(1):e0210513
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: FlexiCyte™ Assay
- Research area: Oncology
The poor prognosis of hepatocellular carcinoma (HCC) has been attributed to a high frequency of tumor metastasis and recurrence even after successful surgical resection. With less than 30% of patients benefiting from curative treatment, alternative treatment regimens for patients with...
View PublicationBromelain Inhibits the Ability of Colorectal Cancer Cells to Proliferate via Activation of ROS Production and Autophagy
-
Tung-Cheng Chang , Po-Li Wei , Precious Takondwa Makondi , et al. ,
-
PLoS One ,
-
2019,
-
14(1):e0210274
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Advanced colorectal cancer (CRC) survival rates are still low despite advances in cytotoxic and targeted therapies. The development of new effective or alternative therapies is therefore urgently needed. Bromelain, an extract of pineapple, was shown to have anticancer effects, but...
View PublicationAspirin Disrupts the Crosstalk of Angiogenic and Inflammatory Cytokines between 4T1 Breast Cancer Cells and Macrophages
-
Hsieh CC, Wang CH ,
-
Mediators of Inflammation ,
-
2018,
-
2018:6380643
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Eukaryotic Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
The tumor microenvironment is rich in multiple cell types that influence tumor development. Macrophages infiltrate tumors, where they are the most abundant immune cell population and secrete a number of cytokines. Aspirin acts as a chemopreventive agent against cancer development....
View PublicationFolate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer
-
Kim M, Pyo S, Kang CH, et al. ,
-
PLoS One ,
-
2018,
-
13(6):e0198347
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in...
View PublicationHeat shock protein 27 influences the anti-cancer effect of curcumin in colon cancer cells through ROS production and autophagy activation
-
Liang HH, Huang CY, Chou CW, et al. ,
-
Life Sci ,
-
2018,
-
209:43-51
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: FlexiCyte™ Assay
- Research area: Oncology
The problem of therapeutic resistance and chemotherapeutic efficacy is tricky and critical in the management of colorectal cancer (CRC). Curcumin is a promising anti-cancer agent. Heat shock protein 27 (HSP27) is correlated with CRC progression and is said to affect...
View PublicationSelf-assembly of tumor-targeting RNA nanoball for carrier-free delivery of therapeutic RNA
-
Han S, Lee JB ,
-
Journal of Industrial and Engineering Chemistry ,
-
2018,
-
64,90-96
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Rolling circle transcription (RCT) has been studied for enhancing the delivery efficiency of therapeutic RNA by densely packing RNA strands with particle formation. This facile fabrication method has been successfully employed to generate RNA nanoballs containing functional short interference RNA....
View PublicationBumetanide attenuates acute lung injury by suppressing macrophage activation
-
Hung CM, Peng CK, Wu CP, Huang KL ,
-
Biochem Pharmacol ,
-
2018,
-
156:60-67
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Leukocytes
- Assay: FlexiCyte™ Assay
- Research area: Immunology
Bumetanide is a potent loop diuretic that acts as an inhibitor of sodium-potassium-chloride cotransporter 2 (NKCC2) and its isoform NKCC1. Although the expression of NKCC2 is limited to the kidney, NKCC1 is widely expressed in various cells, where it participates...
View PublicationViable acrosome-intact human spermatozoa in the ejaculate as a marker of semen quality and fertility status
-
Egeberg Palme DL, Rehfeld A, Bang AK, et al. ,
-
Hum Reprod ,
-
2018,
-
33(3):361-371
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Sperm Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Study question: Is it possible, in an unbiased and clinical relevant way, to determine the number of viable acrosome-intact human spermatozoa in ejaculates and to use this as a measure of fertility chances? Summary answer: Image cytometry enables easy and...
View PublicationTetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungalactivity
-
Bublitz M, Kjellerup L, Cohrt KO, et al. ,
-
PLoS One ,
-
2018,
-
13(1):e0188620
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Yeast Cells
- Assay: Cell Count & Viability Assay, FlexiCyte™ Assay
- Research area: Cell Biology
We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate...
View PublicationSize-Controllable Enzymatic Synthesis of Short Hairpin RNA Nanoparticles by Controlling the Rate of RNA Polymerization
-
Kim H, Kim D, Jeong J, et al. ,
-
Polymers ,
-
2018,
-
10(6), 589
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Thanks to a wide range of biological functions of RNA, and advancements in nanotechnology, RNA nanotechnology has developed in multiple ways for RNA-based therapeutics. In particular, among RNA engineering techniques, enzymatic self-assembly of RNA structures has gained great attention for...
View PublicationInhibitory effects of rosmarinic acid on pterygium epithelial cells through redox imbalance and induction of extrinsic and intrinsic apoptosis
-
Chen YY, Tsai CF, Tsai MC, et al. ,
-
Exp Eye Res ,
-
2017,
-
160:96-105
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Pterygium is a common tumor-like ocular disease, which may be related to exposure to chronic ultraviolet (UV) radiation. Although the standard treatment for pterygium is surgical intervention, the recurrence rate of pterygium is high when no effective inhibitory drug is...
View PublicationEvidence of oxidative stress and mitochondrial dysfunction in spinocerebellar ataxia type 2 (SCA2) patient fibroblasts: Effect of coenzyme Q10 supplementation on these parameters.
-
Cornelius N, Wardman JH, Hargreaves IP, et al. ,
-
Mitochondrion ,
-
2017,
-
34:103-114
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Spinocerebellar ataxia type 2 (SCA2) is a rare neurodegenerative disorder caused by a CAG repeat expansion in the ataxin-2 gene. We show increased oxidative stress, abnormalities in the antioxidant system, changes in complexes involved in oxidative phosphorylation and changes in...
View PublicationPoly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption.
-
Ha JS, Lee JS, Jeong J, et al. ,
-
J Control Release ,
-
2017,
-
250:27-35
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) can be used for the specific disruption of a target gene to permanently suppress the expression of the protein encoded by the target gene. Efficient delivery of the system to...
View PublicationSynthesis of multi-functional DNA nanosphere barcode system for direct cell detection
-
H Sangwoo, Jae Sung Lee and Jong Bum Lee ,
-
Nanoscale ,
-
2017,
-
9(37):14094-14102
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Nucleic acid-based technologies have been applied to numerous biomedical applications. As a novel material for target detection, DNA has been used to construct a barcode system with a range of structures. This paper reports multi-functionalized DNA nanospheres (DNANSs) by rolling...
View PublicationIdentification of Prognostically Relevant Chromosomal Abnormalities in Routine Diagnostics of MultipleMyeloma Using Genomic Profiling.
-
Kjeldsen E ,
-
Cancer Genomics Proteomics ,
-
2016,
-
13(2):91-127
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Leukocytes
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Application of image cytometry to characterize heterologous lipid flippases in yeast.
-
Jensen MS, Costa SR, Theorin L, et al. ,
-
Cytometry A ,
-
2016,
-
;89(7):673-80
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Yeast Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Gene Knockdown in Human Rhinovirus 1B Using 2′-OMe-modified siRNAs Results in the Reactivation of theInterferon Response
-
Xie GC, Zhang Q, Pang LL, et al. ,
-
Biomed Environ Sci ,
-
2016,
-
29(2):137-42
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Immunology
Cell-Based Lipid Flippase Assay Employing Fluorescent Lipid Derivatives.
-
Jensen MS, Costa S, Günther-Pomorski T, López-Marqués RL. ,
-
Methods Mol Biol. ,
-
2016,
-
1377:371-82.
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Yeast Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Generation of siRNA Nanosheets for Efficient RNA Interference
-
Kim H, Lee JS, Lee JB ,
-
Sci Rep ,
-
2016,
-
6:25146
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Self-assembled DNA-Guided RNA Nanovector via Step-wise Dual Enzyme Polymerization (SDEP) for Carrier-free siRNA Delivery
-
Park Y, Hyejin Kim, and Jong Bum Lee ,
-
ACS Biomater. Sci. Eng ,
-
2016,
-
2(4):616-624
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders
-
Fernandez-Guerra P, Lund M, Corydon TJ, et al. ,
-
JIMD Rep ,
-
2016,
-
27:17-26
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Cell Count & Viability Assay, Cell Vitality Assay, FlexiCyte™ Assay, Mitochondrial Potential Assay
- Research area: Cell Biology
hiPS-Mesenchymal Stem Cellss differentiation towards fibroblasts on a 3D ECM mimicking scaffold.
-
Xu R, Taskin MB, Rubert M, et al. ,
-
Sci Rep ,
-
2015,
-
6,097222222
- Instrument: NucleoCounter® NC-3000™
- Cell Type: MSCs
- Assay: FlexiCyte™ Assay
- Research area: Stem Cell Research
Guanidine modified polyethyleneimine-g-polyethylene glycol nanocarriers for long interfering RNA (liRNA) based advanced anticancer therapy
-
Sajeesh S, Jeong Yong Choe, Tae Yeon Leea and Dong-ki Lee. ,
-
J. Mater. Chem. B ,
-
2015,
-
3(2):207-216
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Dual targeting NG2 and GD3A using Mab-Zap immunotoxin results in reduced glioma cell viability in vitro.
-
Higgins SC, Fillmore HL, Ashkan K, et al. ,
-
Anticancer Res. ,
-
2015,
-
35(1):77-84.
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Colorectal cancer stem cell and chemoresistant colorectal cancer cell phenotypes and increased sensitivity toNotch pathway inhibitor.
-
Huang R, Wang G, Song Y, et al. ,
-
Mol Med Rep ,
-
2015,
-
12(2):2417-24
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
ALPPL2 Aptamer-Mediated Targeted Delivery of 5-Fluoro-2′-Deoxyuridine to Pancreatic Cancer.
-
Dua P, S S, Kim S, Lee DK. ,
-
Nucleic Acid Ther. ,
-
2015,
-
25(4):180-7
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Hyperphosphatemia induces protective autophagy in endothelial cells through the inhibition of Akt/mTOR signaling
-
Hsu YJ, Hsu SC, Huang SM, et al. ,
-
J Vasc Surg ,
-
2015,
-
62(1):210-221
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: Annexin V Assay, FlexiCyte™ Assay
- Research area: Cell Biology
Carbon dots prepared from ginger exhibiting efficient inhibition of human hepatocellular carcinoma cells
-
Li CL, Chung-Mao Ou,a Chih-Ching Huang, et al. ,
-
J. Mater. Chem. B ,
-
2014,
-
2(28):4564-4571
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Long dsRNA-mediated RNA Interference and Immunostimulation: A Targeted Delivery Approach using Polyethyleneimine based Nano-Carriers
-
Sajeesh S, Lee TY, Hong SW, et al. ,
-
Mol Pharm ,
-
2014,
-
11(3):872-84
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Efficient intracellular delivery and multiple-target gene silencing triggered by tripodal RNA based nanoparticles: a promising approach in liver-specific RNAi delivery.
-
Sajeesh S, Lee TY, Kim JK, et al. ,
-
J Control Release ,
-
2014,
-
196:28-36
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
Comparison of three thiol probes for determination of apoptosis-related changes in cellular redox status.
-
Skindersoe ME, Kjaerulff S. ,
-
Cytometry A ,
-
2014,
-
85(2):179-87
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, FlexiCyte™ Assay, Mitochondrial Potential Assay
- Research area: Cell Biology
Resveratrol Inhibits Alpha-Melanocyte-Stimulating Hormone Signaling, Viability, and Invasiveness in Melanoma Cells
-
Chen YJ, Chen YY, Lin YF, et al. ,
-
Evid Based Complement Alternat Med. ,
-
2013,
-
2013:632121
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Alkaline Phosphatase ALPPL-2 Is a Novel Pancreatic Carcinoma-Associated Protein
-
Dua P, Kang HS, Hong SM, et al. ,
-
Cancer Res. ,
-
2013,
-
73(6):1934-45
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Sunitinib and SU11652 inhibit acid sphingomyelinase, destabilize lysosomes and inhibit multidrug resistance
-
Ellegaard AM, Groth-Pedersen L, Oorschot V, et al. ,
-
Mol Cancer Ther. ,
-
2013,
-
12(10):2018-30
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Oncology
Secondary Coenzyme Q10 deficiency and oxidative stress in cultured fibroblasts from patients with Riboflavin Responsive Multiple Acyl-CoA Dehydrogenation Deficiency
-
Cornelius N, Byron C, Hargreaves I, et al. ,
-
Hum Mol Genet ,
-
2013,
-
22(19):3819-27
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
RNA Interference-Mediated Simultaneous Silencing of Four Genes Using Cross-Shaped RNA
-
Lee TY, Chang CI, Lee D, et al. ,
-
Mol Cells ,
-
2013,
-
35(4):320-6
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: FlexiCyte™ Assay
- Research area: Cell Biology
RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells
-
Oestergaard VH, Pentzold C, Pedersen RT, et al. ,
-
DNA Repair ,
-
2012,
-
11(11):892-905
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Annexin V Assay, Cell Cycle Assay, FlexiCyte™ Assay
- Research area: Cell Biology
Oxidative stress is linked to ERK1/2-p16 signaling-mediated growth defect in ATM-deficient astrocytes.
-
Kim J, Wong PK ,
-
J Biol Chem ,
-
2009,
-
284(21)
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: FlexiCyte™ Assay
- Research area: Neurobiology