Fluorokines™ - A Better Fluorescent-Labeled Protein Solution

Fluorokines – Direct Detection of Target Molecules by Flow Cytometry

R&D Systems Fluorokines are fluorescent-labeled recombinant proteins that allow target molecules to be directly stained and detected by flow cytometry. Our catalog includes Fluorokines that are designed for evaluating the expression of specific chimeric antigen receptors on CAR-T or CAR-NK cells, Fluorokines for detecting cells expressing specific immune checkpoint receptors, as well as Fluorokines for assessing SAR-CoV-2 reactive ACE-2+ cells or SARS-CoV-2-related immune responses.

Fluorokines are made by conjugating the bioactive protein to the fluorophore via amine labeling, and the resulting fluorescent-labeled protein is then rigorously tested to ensure consistent labeling of each lot. The entire manufacturing process is controlled to reduce lot-to-lot variability and ensure a consistent F/P ratio. Additionally, the specificity of each protein is quality control tested using flow cytometry to confirm that the Fluorokine stains the appropriate target.

Benefits of Using Fluorescent-labeled Proteins for Detecting Target Molecules

• Direct detection. Direct detection of a target molecule with a Fluorokine is highly specific and eliminates background staining that may occur by indirect detection using an epitope-tagged target antigen and a fluorophore-labeled secondary antibody.
• Reduced processing time. Target detection requires only a single processing step as Fluorokines eliminate the need for a secondary antibody.
• Conjugated to Alexa Fluor^® dyes. Fluorescent-labeled proteins are conjugated to Alexa Fluor dyes, which offer more intense fluorescence than other spectrally similar fluorophore labels and excellent photostability.
• 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 is tested side-by-side with previous lots to ensure high lot-to-lot consistency.

 

Fluorokines for Chimeric Antigen Receptor Detection

The ability to evaluate the expression of a chimeric antigen receptor (CAR) following T cell or natural killer (NK) cell transduction is a critical step in the production of CAR-T or CAR-NK cells for cancer immunotherapy. Using fluorescent-labeled proteins to evaluate CAR expression is highly specific, reduces processing time, and eliminates background staining that may occur by indirect detection of the CAR using an epitope-tagged target antigen and a fluorophore-labeled secondary antibody.

Fluorescent-labeled Proteins for CAR-T or CAR-NK Cell Detection

As directly detecting a CAR with a labeled target antigen is currently the method of choice for evaluating CAR expression, we are diligently working to expand our selection of Fluorokines for CAR-T and CAR-NK cell research. As an alternative method, we also offer Avi-tag biotinylated proteins, which can be detected using fluorochrome-labeled streptavidin. Additionally, if you are unable to find either the fluorescent-labeled or biotinylated protein that you need on our website, our Custom Protein Services team can work with you to create a customized protein solution to meet your specific research needs.

Utilizing Fluorokines Fluorescent-labeled Proteins to Detect CAR-T 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 determined 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 (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 (Catalog # ATM9269) or a Biotinylated Monoclonal Anti-FMC63 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 (Catalog # FAB3832N), PerCP-conjugated Mouse Anti-Human CD45 (Catalog # FAB1430C), PE-conjugated Rabbit Anti-Human CD56/NCAM-1 (Catalog # FAB24086P), Alexa Fluor^® 405-conjugated Mouse Anti-Human CD8 (Catalog # FAB1509V), PE-Cy7-CD4, and Alexa Fluor^® 594-conjugated Mouse Anti-Human CD62L/L-Selectin (Catalog # FAB9787T), along with the Fluorokine, Recombinant Human CD19 Fc Chimera Atto 647N Protein (Catalog # ATM9269). Cells were initially gated on singlets and live cells.

Specificity, Bioactivity, and Lot-to-Lot Consistency Testing of R&D Systems Fluorokines for CAR-T Cell Research

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 (Catalog # ATM193) or (B) unstained and detected by flow cytometry. (C) Recombinant Human APRIL/TNFSF13 (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 (Catalog # ATM193; red, green, orange lines) or unlabeled Recombinant Human BCMA Fc Chimera (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.

 

Fluorokines for Immune Checkpoint Ligands

Immune checkpoint molecules deliver signals that control the activities of different immune cell types and represent some of the most promising targets for immuno-oncology research. Utilize our new Alexa Fluor^®-labeled immune checkpoint ligands to easily sort or detect cells expressing specific immune checkpoint receptors. Fluorescent-labeled immune checkpoint ligands bind to cells expressing their corresponding receptors in a highly specific manner, allowing the direct detection of these cells by flow cytometry.

Alexa Fluor^®-labeled Immune Checkpoint Ligands

Utilizing Fluorokines Fluorescent-labeled Proteins 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 (Catalog # AFR9049). (B) Streptavidin-coated beads conjugated to Biotinylated Anti-Human B7-1/CD80 Monoclonal Antibody (Catalog # BAM1402) were stained with the indicated concentrations of Recombinant Human B7-1/CD80 His-tag Alexa Fluor^® 647 Protein (Catalog # AFR9050).

Fluorokines for SARS-CoV-2 Spike Proteins

The SARS-CoV-2 Spike protein is a key factor regulating viral attachment and fusion of the viral and host cell membranes, and therefore, it is a primary target for therapeutic research. To help facilitate this research, Bio-Techne now offers a wide selection of Alexa Fluor^®-labeled, bioactive SARS-CoV-2 Spike proteins, including the full ectodomain SARS-CoV-2 Spike protein, the Spike protein receptor binding domain (RBD), and Alexa Fluor-labeled alpha, beta, gamma, delta, kappa, and omicron Spike protein variants. These proteins can be used to directly detect SARS-CoV-2 reactive ACE-2+ cells or to investigate SARS-CoV-2-related immune responses. All fluorescent-labeled Spike proteins are tested to ensure that they detect ACE-2 by flow cytometry.

Alexa Fluor^®-labeled SARS-CoV-2 Spike Proteins

Utilizing Fluorokines Fluorescent-labeled Proteins for SARS-CoV-2 Research

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 Recombinant SARS-CoV-2 Spike (GCN4-IZ) His-tag Alexa Fluor^® 488 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 (Catalog # AFG10561) or (B) unstained. Nearly all cells were positive for ACE-2 expression compared to the unstained control.

Custom Protein Services

If you are unable to find the fluorescent-labeled protein that you need on our website, contact our Custom Protein Services team. They will work with you to create a customized protein solution to meet your specific research needs.

 

Bulk Proteins

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Fluorokine Related Resources

Fluorokine Related Research Areas

Acute Lymphoblastic Leukemia

B Cells

B Cell Markers

Lymphoma

Hematologic Cancers

Multiple Myeloma

Video Transcript

R&D Systems fluorescent-labeled proteins allow for specific detection of a chimeric antigen receptor on cells by flow cytometry. Once genetic engineering is performed instructing the cells to express a specific CAR, the cells are cultured allowing for the expression of the CAR on the cell surface. A fluorescently labeled protein that binds to the CAR can be used to determine the cells that are positive by flow cytometry. R&D Systems fluorescent proteins are made by conjugating a fluorescent dye to a bioactive recombinant protein. These proteins are purified and QC tested to ensure bioactivity and lot-to-lot consistency. Advantages of using fluorescently labeled proteins include reduced processing time, no secondary antibody step, and reduced potential for variability and unwanted background staining. Streamline your car expression workflows with R&D Systems, the number one most trusted brand in recombinant proteins.