R&D Systems™ Fluorokines or fluorescent-labeled recombinant proteins can be used to directly stain and detect target molecules by flow cytometry. Our catalog includes Fluorokines designed to simplify the detection of chimeric antigen receptors on CAR-T or CAR-NK cells, Fluorokines for detecting the expression of immune checkpoint receptors, and fluorescent-labeled SARS-CoV-2 Spike proteins for evaluating SARS-CoV-2-binding cells or SARS-CoV-2-induced immune responses. Direct staining with a Fluorokine reduces processing time and eliminates background staining that may occur by indirect detection using a secondary antibody.
Fluorokine proteins are conjugated to a fluorophore via amine labeling, and rigorous in-house testing is performed to confirm consistent labeling of each lot. The entire Fluorokine manufacturing process is strictly controlled to ensure minimal lot-to-lot variability and a consistent F/P ratio. Additionally, the specificity of each Fluorokine is tested to confirm that it stains the appropriate target by flow cytometry.
While we are currently working on expanding our selection of Fluorokines, if you don’t see the fluorescent-labeled protein that you need on our website, please contact us. Our Custom Protein Services team will be happy to see if we can assist you with a customized protein solution that will meet your specific research needs. Alternatively, please browse our wide selection of Avi-tag and amine-labeled biotinylated proteins, which can be used to stain target molecules and then detected using fluorochrome-conjugated streptavidin.
Technique Talk:
An Improved Way to Detect CAR-T Cells and SARS-CoV-2-Binding Cells Using Fluorokines
Advantages of Fluorescent-labeled Proteins for Detecting Target Molecules
• Direct detection. Fluorokines are highly specific and allow target molecules to be directly detected, eliminating the possibility of background staining that may occur by indirect detection using a secondary antibody.
• Conjugated to Alexa Fluor® Dyes. Fluorescent-labeled proteins are conjugated to Alexa Fluor® dyes, which offer intense fluorescence and excellent photostability.
• Reduced Processing Time. Target detection requires only a single processing step as Fluorokines eliminate the need for staining with a secondary antibody.
• High Levels of Bioactivity. Fluorescent-labeled proteins are made using R&D Systems highly active, unlabeled recombinant proteins.
• High lot-to-lot consistency. Each new lot of a Fluorokine protein is tested side-by-side with previous lots to ensure high lot-to-lot consistency.
Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
Fluorokines for Chimeric Antigen Receptor Detection
The ability to evaluate the expression of a chimeric antigen receptor (CAR) following retroviral or lentiviral transduction of a patient’s or donor’s T or natural killer (NK) cells is an important step in the production of CAR-T or CAR-NK cells for cancer immunotherapy. Using fluorescent-labeled proteins, CAR+ cells can be directly stained and detected by flow cytometry, making it easy to evaluate the percentage of transduced cells. Fluorokines are highly specific, reduce processing time, and eliminate background staining that may occur with indirect detection using a secondary antibody.
Fluorokines Can Be Used to Directly Assess CAR Expression on CAR-T or CAR-NK Cells
Demonstration of the Utility of a Fluorokine for Evaluating CAR Expression. (A) CAR-T cell therapy is based on the principle that T cells removed from a patient or donor can be genetically engineered to express a specific chimeric antigen receptor (CAR). Once these CAR-T cells are infused back into the patient, the CAR will bind to its specific target antigen on the surface of the patient’s tumor cells, activating the T cells, and allowing them to attack and destroy the tumor cells. (B) The ability to evaluate CAR expression following T cell transduction is an important step in the production of CAR-T cells. T cells expressing the CAR can be directly stained using a Fluorokine (target antigen) and the percentage of CAR-expressing cells can be detected by flow cytometry. (C) CD4+CD8+ T cells were transduced with a hCD19-CAR (left) or not transduced (right) and then cultured for 11 days. Cells were stained with a PE-Cy7-CD4 and Recombinant Human CD19 Fc Chimera Atto 488 Protein (R&D Systems, Catalog # ATJ9269), and detected by flow cytometry.
Detection of CD19-CAR Expression with a Recombinant Human CD19 Atto647N Fluorokine or an Anti-Idiotype FMC63 scFv Antibody Gives Comparable Results
Detection of CD19-CAR T Cells with a Recombinant Human CD19 Atto647N Fluorokine or an Anti-FMC63 scFv Monoclonal Antibody Gives Comparable Results. Edited T cells were assessed for CAR incorporation via flow cytometry using either the Recombinant Human CD19 Fc Chimera Atto 647N Protein (R&D Systems, Catalog # ATM9269) or a Biotinylated Anti-FMC63 Monoclonal Antibody followed by PE-conjugated Streptavidin. Staining results show comparable detection of the hCD19-CAR using either the fluorescent-labeled CD19 protein or the anti-idiotype FMC63 scFv antibody.
Fluorokines Are Compatible with Antibody Panels for Multi-Color Flow Cytometry
Fluorokines can be used with Fluorochrome-conjugated Antibodies to Characterize CD19-CAR-T Cells by Multi-Color Flow Cytometry. CD4+CD8+ T cells transduced with a hCD19-CAR were stained with the following panel of monoclonal antibodies: Alexa Fluor® 700-conjugated Mouse Anti-Human CD14 (R&D Systems, Catalog # FAB3832N), PerCP-conjugated Mouse Anti-Human CD45 (R&D Systems, Catalog # FAB1430C),PE-conjugated Rabbit Anti-Human CD56/NCAM-1 (R&D Systems, Catalog # FAB24086P), Alexa Fluor® 405-conjugated Mouse Anti-Human CD8 (R&D Systems, Catalog # FAB1509V), PE-Cy7-CD4, and Alexa Fluor® 594-conjugated Mouse Anti-Human CD62L/L-Selectin (R&D Systems, Catalog # 9787T) , along with the Fluorokine, Recombinant Human CD19 Fc Chimera Atto 647N Protein (R&D Systems, Catalog # ATM9269). Cells were initially gated on singlets and live cells.
R&D Systems Fluorokines Undergo Rigorous Testing to Ensure Specificity, Bioactivity, and Lot-to-Lot Consistency
Analysis of the Specificity, Bioactivity, and Lot-to-Lot Consistency of Recombinant Human BCMA/TNFRSF17 Fc Chimera Atto 647N Protein. Fluorescent beads conjugated to an anti-Human BCMA Monoclonal Antibody were stained with (A) Recombinant Human BCMA/TNFRSF17 Fc Chimera Atto 647N Protein (R&D Systems, Catalog # ATM193) or (B) unstained and detected by flow cytometry. (C) Recombinant Human APRIL/TNFSF13 (R&D Systems, Catalog # 5860-AP) was immobilized at 0.1 ug/mL, 100 ul/well, and the indicated concentrations of three independent lots of Recombinant Human BCMA Fc Chimera Atto 647N (R&D Systems, Catalog # ATM193; red, green, orange lines) or unlabeled Recombinant Human BCMA Fc Chimera (R&D Systems, Catalog # 193-BC; blue line) were added. The data demonstrates consistent bioactivity between the fluorescent-labeled and unlabeled proteins and lot-to-lot consistency in the bioactivity of the three different lots of the fluorescent-labeled protein.
Detection of CD19-CAR-T Cells with Recombinant Human CD19 Fc Chimera Atto 647N Protein. CD4+CD8+ T cells were either (A) transduced with a hCD19-CAR or (B) not transduced, and then cultured for 11 days. Cells were stained with a PE-Cy7-CD4 and Recombinant Human CD19 Fc Chimera Atto 647N Protein (R&D Systems, Catalog # ATM9269) and detected by flow cytometry.
Fluorokines for Immune Checkpoint Ligands
Immune checkpoint molecules regulate the activation and functions of different immune cell types and represent some of the most highly investigated targets for cancer immunotherapy. Take advantage of our fluorescent-labeled immune checkpoint ligands to easily identify or sort cells expressing the corresponding immune checkpoint receptors. Fluorescent-labeled ligands allow cells expressing their cognate receptors to be stained in a single step and detected by flow cytometry.
Fluorescent-labeled Immune Checkpoint Ligands - Products by Molecule
Fluorokines for Immuno-Oncology Research
Using Fluorokines to Discover Immune Checkpoint Interactions for Immunotherapy. (A) Typical interactions of immune checkpoint ligands and receptors involve an antigen presenting cell expressing a ligand like PD-L1 or PD-L2 and a T cell expressing a receptor like PD-1. Binding of receptor and ligand initiates a signaling cascade to either inhibit or activate cellular responses. In the case of PD-1 binding, T cell activity is inhibited. (B) Using Immune Checkpoint Fluorokines aid researchers in studying immune responses, sort immune cells and test the efficacy of new immunotherapies that disrupt Checkpoint ligand-receptor interactions.
Analysis of the Specificity of the Recombinant Human PD-L1/B7-H1 and B7-1/CD80 His-tag Alexa Fluor® 647 Proteins
Analysis of the Specificity of the Recombinant Human PD-L1/B7-H1 and B7-1/CD80 His-tag Alexa Fluor® 647 Proteins. (A) Streptavidin-coated beads conjugated to Biotinylated Anti-Human PD-L1/B7-H1 Monoclonal Antibody were stained with the indicated concentrations of Recombinant Human PD-L1/B7-H1 His-tag Alexa Fluor® 647 Protein (R&D Systems, Catalog # AFR9049). (B) Streptavidin-coated beads conjugated to Biotinylated Anti-Human B7-1/CD80 Monoclonal Antibody (R&D Systems, Catalog # BAM1402) were stained with the indicated concentrations of Recombinant Human B7-1/CD80 His-tag Alexa Fluor® 647 Protein (R&D Systems, Catalog # AFR9050).
Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
Fluorokines for SARS-CoV-2 Spike Proteins
The SARS-CoV-2 Spike protein mediates binding to the host cell ACE-2 receptor, membrane fusion, and viral entry, making it a primary target for potential COVID-19 therapeutics. Our line of fluorescent-labeled SARS-CoV-2 Spike proteins was developed to make it easier for researchers to identify SARS-CoV-2-binding cells, screen for antibodies or small molecules that block the Spike-ACE-2 protein interaction, and investigate SARS-CoV-2-related immune responses. Our catalog includes fluorescent-labeled versions of the SARS-CoV-2 Spike protein full ectodomain and receptor binding domain (RBD), along with fluorescent-labeled SARS-CoV-2 Spike proteins for the alpha, beta, gamma, delta, kappa, and omicron SARS-CoV-2 variants. All fluorescent-labeled Spike proteins are tested to ensure that they detect ACE-2 by flow cytometry.
Fluorescent-labeled SARS-CoV-2 Spike Proteins and ACE-2 - Products by Molecule
Discover SARS-CoV-2 Interactions and Immune Responses Using Fluorokines
Utilizing Ligand and Receptor-conjugated Fluorokines for SARS-CoV-2 Research. (A) The interaction between SARS-CoV-2 Spike proteins and the host cell receptor ACE-2 is a prime target for COVID-19 therapeutics due to its functions in membrane fusion and viral entry into cells. (B, C) Fluorescent-labeled Spike proteins or ACE-2 receptors allow for the identification of SARS-CoV-2 binding cells and can be used as tools for investigating immune responses or identifying molecules that may disrupt SARS-CoV-2 Spike and ACE-2 interactions.
Assessing ACE-2+ Cells Using Alexa Fluor® 488-Labeled SARS-CoV-2 Spike Protein
Alexa Fluor® Spike Proteins Detect ACE-2 Expression on HEK293 Cells. HEK293 cells transfected with human ACE-2 were stained with (A) 1 ug/mL (100 uL/well) Recombinant SARS-CoV-2 Spike (GCN4-IZ) His-tag Alexa Fluor® 488 Protein (R&D Systems, Catalog # AFG10561) or (B) unstained. Nearly all cells were positive for ACE-2 expression compared to the unstained control.
Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
Custom Protein Services
If you don’t see the fluorescent-labeled protein that you need listed on our website, contact our Custom Protein Services team. From scratch protein development to customizing a protein from our catalog, our custom team will work with you to create the protein that meets your experimental needs.
Immune Checkpoint Targets Wall Poster
Immune checkpoint molecules play a central role in regulating the activities of different immune cell types and represent some of the most promising targets for immuno-oncology research. Use our new wall poster to explore the current and emerging immune checkpoint molecules being investigated as potential targets for cancer immunotherapy.
Fluorokine Related Resources
Avi-tag Biotinylated Proteins
Avi-tag Biotinylated Proteins
If you can’t find the fluorescent-labeled protein that you need on our website, explore our wide selection of Avi-tag biotinylated proteins. Avi-tag biotinylated proteins can be used with fluorochrome-labeled streptavidin to detect chimeric antigen receptors, immune checkpoint receptors, or SARS-CoV-2-binding cells.
Evaluating CAR Expression on CAR-T Cells Using FluorokinesTM
Evaluating CAR Expression on CAR-T Cells Using FluorokinesTM
One of the current challenges in the field of CAR-T cell therapy is the lack of a simple assay to easily detect and quantify CAR expression following T cell transduction. Read this application note to learn about the advantages of using R&D Systems Fluorokines fluorescent-labeled proteins to directly stain and detect CAR+ cells by flow cytometry.
Flow Cytometry-Validated Antibodies for Identifying Immune Cell Types
Flow Cytometry-Validated Antibodies for Identifying Immune Cell Types
The Bio-Techne family brands, R&D Systems and Novus Biologicals, are committed to providing the highest quality antibodies to support your research. Easily identify and characterize different immune cell types using our wide selection of flow cytometry-validated antibodies.
Fluorokine Related Research Areas
Immune Cell Therapy
Immune Cell Therapy
Harness the power of immune cells to attack specific tumor cell populations. Isolate, expand, engineer, and characterize your cells with our premium reagents, services, and instruments. Our flexible and innovative tools will help to simplify your workflow at every step of the manufacturing process.
Immune Checkpoint Proteins
Immune Checkpoint Proteins
Browse our large selection of proteins for studying current and emerging immune checkpoint molecules. In addition to the common targets such as PD-1, PD-L1, and CTLA-4, we offer many lesser-known immune checkpoint proteins that exhibit similar T cell co-inhibitory effects.
Proteins for Coronavirus Research
Proteins for Coronavirus Research
As the COVID-19 pandemic evolves and new SARS-CoV-2 mutant variants are identified, we are striving to provide researchers with the tools that they need to more fully understand this virus. Bio-Techne offers over 100 different SARS-CoV-2 Spike proteins, including multiple different versions of the Alpha, Beta, Gamma, Delta, Omicron, and IHU Spike protein variants.
Proteins for Immune Cell Culture
Achieve robust, reproducible immune cell cultures with R&D Systems™ proteins. Our proteins are rigorously tested to ensure that they will provide superior performance and lot-to-lot consistency, so you can have confidence in their ability to promote optimal immune cell expansion and differentiation with minimal variability between cultures.
