TLR Reporter Bioassay

Simple Bioassay, Quality Results

The human immune system responds to conserved damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, respectively) via pattern-recognition receptors (PRRs). One such family of PRRs is the Toll-like receptor (TLR) family. In humans, there are 10 known TLRs, which enable the immune system to sense and respond to damage and danger signals. Modulation of the TLR system, either by agonism or antagonism, has the potential to alter immune and inflammatory responses. Blockade of TLRs may inhibit inflammatory responses while TLR agonists may promote immune responses against pathogens or cancer.

Current methods for assessing the activity of TLR modulators typically rely on primary monocyte-derived macrophages and measurement of functional endpoints, such as cytokine production. These assays are laborious and highly variable due to their reliance on donor cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in a quality-controlled setting.

The TLR Bioassay is a bioluminescent reporter cell-based assay used to measure the potency and stability of TLR agonists.

Bioassay Components and Product Formats

The TLR Reporter Bioassay consists of one genetically engineered cell line:

TLR Bioassay Cells: A monocytic cell line with a stably integrated NanoLuc® (NL) luciferase reporter driven by TLR pathway-dependent response elements.

Thaw-and-Use Kits (1X, 5X):

Include TLR Bioassay Cells, cell culture media and assay reagents
Recommended for routine use with customer-defined agonist
Available in 1X and 5X sizes

Cell Propagation Model format (CPM; Cat.# GA1322):

Includes the TLR Bioassay Cells (CPM); these are cryopreserved cells that can be thawed, propagated and banked for long-term use.
Use with Bio-Glo-NL™ Luciferase Assay System

Cell Bank format (Cat.# GA6010):

Available for customers who are planning to expand and bank their CPM purchase, but they would like to use the cell expansion service offered by Promega.

Workflow

Standardized Reagents Improve Results

Thaw-and-use format provides significant time and labor savings over traditional methods while reducing variability.
Easily amenable to high-throughput workflows.

Assay Principle

The TLR Reporter Bioassay measures the potency and stability of agonists designed to activate TLR signaling.

**Representation of the TLR Reporter Bioassay.** Treatment with a TLR agonist results in promoter-driven luminescence that can be detected and quantified using Bio-Glo-NL™ Reagent.

TLR Ligand Specificity.

TLR	PAMPs	DAMPs
TLR1/TLR2	Triacylated lipoproteins (e.g., Pam3CSK4) Peptidoglycans	HMGB1 HSP60, 70, Gp76 Peptidoglycans
TLR2/TLR6	Diacylated lipoproteins (e.g., FSL-1) LTA Zymosan	HMGB1 HSP60, 70, Gp76 Peptidoglycans
TLR3	dsRNA (e.g., Poly (I:C))	mRNA tRNA
TLR4	Lipopolysaccharide (LPS)	HMGB1 Peptidoglycans HSP22, 60, 70, 72, Gp96
TLR5	Flagellin
TLR7	ssRNA Imidazoquinolines (e.g., R848) Guanosine analogs	ssRNA
TLR8	ssRNA Imidazoquinolines (e.g., R848)	ssRNA
TLR9	CpG DNA CpG ODNs	CpG DNA CpG ODNs
TLR10	Profilin-like protein

Application Data: Targeting TLR Pathways

The TLR Bioassay luminescent readout can be used in a wide variety of important applications where modulating the TLR system, either by agonism or antagonism, is critical in understanding immune and inflammatory responses. Applications include TLR signaling for vaccine adjuvant development, antitumor activity and host-pathogen interactions.

19358ma — **TLR4 agonism measured using the TLR Reporter Bioassay.** The thaw-and-use TLR Bioassay was used to compare the TLR4 agonism between LPS, MPLA and CRX-527 to show varying levels of signaling for adjuvant ranking.

19359ma — **TLR signaling for enhancing antitumor effects.** R848 (resiquimod) is a synthetic imidazoquinoline compound recognized by TLR7 and TLR8 to modify the immune response from immune cells, like macrophage.

19360ma — **Bacterial pathogen-associated molecular patterns (PAMPs) trigger TLR signaling.** Lipoteichoic acid (LTA) is an immunogenic PAMP found in the cell wall of many Gram-positive bacteria and recognized by TLR2. LTA from *Staphylococcus aureus* was used to generate the data.

Protocols

Complete Protocol

TLR Bioassay Technical ManualPDF (1931 KB) – English

TLR Bioassay Cells, Propagation Model Technical ManualPDF (1987 KB) – English

What's in the box?

Item	Part #	Size
TLR Bioassay Cells	GA132A	1 × 0.5ml
Fetal Bovine Serum	J121A	1 × 4ml
RPMI 1640 Medium	G708A	1 × 36ml
Bio-Glo-NL™ Luciferase Assay Buffer	J308A	1 × 10ml
Bio-Glo-NL™ Luciferase Assay Substrate	J309A	1 × 200μl

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use; (2) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo-NL™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 8,809,529, European Pat. No. 2635582, Japanese Pat. No. 5889910 and other patents and patents pending.

U.S. Pat. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.

Product cannot be used for proficiency testing.

Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.

What's in the box?

Item	Part #	Size
TLR Bioassay Cells	GA132A	5 × 0.5ml
Fetal Bovine Serum	J121A	5 × 4ml
RPMI 1640 Medium	G708A	5 × 36ml
Bio-Glo-NL™ Luciferase Assay Buffer	J308A	5 × 10ml
Bio-Glo-NL™ Luciferase Assay Substrate	J309A	5 × 200μl

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use; (2) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to propagate, modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells. In addition, recipient must use Bio-Glo-NL™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. No. 8,809,529, European Pat. No. 2635582, Japanese Pat. No. 5889910 and other patents and patents pending.

U.S. Pat. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.

Product cannot be used for proficiency testing.

Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.

What's in the box?

Item	Part #	Size
TLR Bioassay Cells (CPM)	GA130A	2 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use; (2) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells except that recipient may propagate and store the cells for long-term use. In addition, recipient must use Bio-Glo-NL™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.

U.S. Pat. No. 8,809,529, European Pat. No. 2635582, Japanese Pat. No. 5889910 and other patents and patents pending.

Product cannot be used for proficiency testing.

Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.

What's in the box?

Item	Part #	Size
TLR Bioassay Cells (CPM)	GA130A	50 × 1ml

SDS

Search for SDS

Certificate of Analysis

Search by lot number

Use Restrictions

For Research Use Only. Not for Use in Diagnostic Procedures.

Storage Conditions

Please see the Protocol for detailed storage conditions.

Patents and Disclaimers

NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RECIPIENT AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE STATEMENT. If the recipient is not willing to accept the conditions of this limited use statement, and the product is unused, Promega will accept return of the unused product and provide the recipient with a full refund.

This product may not be further sold or transferred by the recipient and may be used only by the recipient, and then only for (1) research use; (2) discovery, development and monitoring of biologic drugs and vaccines; (3) quality assurance testing of biologic drugs and vaccines; and (4) product release assays for biologic drugs and vaccines. No other commercial use is allowed. "Commercial use" means any and all uses of this product by recipient for monetary or other consideration, including providing a service, information or data to unaffiliated third parties, and resale of this product for any use. Recipient has no right to modify, derivatize, genetically engineer or otherwise create variations of the cells or genes stably transfected within the cells except that recipient may propagate and store the cells for long-term use. In addition, recipient must use Bio-Glo-NL™ Luciferase Assay System purchased from Promega Corporation for all luminescence assays using this product or contact Promega to obtain a license for use of this product with reagents other than Promega's. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this agreement shall be governed under the laws of the State of Wisconsin, USA.

U.S. Pat. Nos. 8,557,970, 8,669,103, 9,777,311, 9,840,730, 9,951,373, 10,233,485, 10,633,690, 10,774,364, 10,844,422, 11,365,436, 11,661,623, 11,667,950; European Pat. Nos. 2456864, 2635595, 2990478, 3181687, 3409764; Japanese Pat. Nos. 6038649, 6155424, 6227615, 6374420, 6539689; and other patents and patents pending.

Product cannot be used for proficiency testing.

Licensed from Lonza Cologne GmbH under U.S. Pat. Nos. 7,700,357, 8,192,990 and 8,003,389, European Pat. Nos. 1297119, 1522587, 1607484 and 1741778 and other pending and issued patents.

Resources

Posters

Other Resources

Similar Products

PD-1/PD-L1 Blockade Bioassay

Measure the ability of biologics to block immune checkpoint signals

J1250, J1255, J1252, J1191, J1195, J1201

Membrane TNFα Target Cells

Assess the mechanism of action of biologics designed to affect TNFα signaling.

J3331, J3335, J3322

ADCP Reporter Bioassay (THP-1)

Measure the potency and stability of antibodies and other biologics with Fc domains that bind and activate FcγRs.

JA9411, JA9415, GA1272, GA6020

SIRPα/CD47 Blockade Bioassay, Fc-Dependent

Measure the potency and stability of biologics that bind and block SIRPα/CD47 interactions.

JA4801, JA4805, JA4802, GA6000, GA6030

TLR Reporter Bioassay

Measure Potency and Stability of Molecules that Modulate TLR Signaling