Phospho-SLP-76 (Ser376) cellular kit HTRF®

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The phospho-SLP-76 (Ser376) kit enables the cell-based quantitative detection of phosphorylated SLP-76 at Ser376, for monitoring T-lymphocyte activation.

Ready-to-use
High sensitivity
Faster and more convenient than ELISA
No-wash

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The phospho-SLP-76 (Ser376) kit enables the cell-based quantitative detection of phosphorylated SLP-76 at Ser376, for monitoring T-lymphocyte activation.

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Overview

This HTRF cell-based assay enables rapid, quantitative detection of SLP-76 phosphorylated on Serine 376. Phospho-SLP-76 creates a scaffold on which key signaling complexes are built, and is a marker of T-lymphocyte activation

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Phospho-SLP-76 (Ser376) assay principle

The Phospho-SLP-76 (Ser376) assay measures SLP-76 when phosphorylated at Ser376. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-SLP-76 (Ser376) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the proteins phosphorylation state under a no-wash assay format.

Phospho-SLP-76 (Ser376) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-SLP-76 (Ser376) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-SLP-76 (Ser376) 1-plate assay protocol

Detection of Phosphorylated SLP-76 (Ser376) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

HTRF assay compared to Western Blot using phospho SLP-76 (Ser376) cellular assays on human Jurkat cells

Jurkat cells were grown in a T175cm2 flask for 2 days in RPMI culture medium supplemented by 10% FBS, at 37°C in 5% CO2 atmosphere. 4.5 mL of cell suspension (13 x 106 cells / ml) were stimulated with anti-CD3 antibody (20µg/mL final concentration during 2.5min). After stimulation, the cells were lysed at RT for 30 min by adding 2.25 mL of 4X supplemented lysis buffer. Neat lysate was then serially diluted in the same supplemented lysis buffer. 16 µL of each dilution were analyzed in parallel by HTRF or by Western Blot.

Cell density optimisation for Phospho SLP-76 (Ser376)

This cell density optimization was carried out using the two plate protocol. Jurkat cells were plated in a 96-well culture plate at 200,000 Kcells or 400Kcells in 25µL/well, in complete RPMI culture medium with 10% FBS. Cell stimulation was done by anti-CD3 Ab at various concentrations for 30 min. Cell lysis was performed using 10 µL of 4X supplemented lysis buffer. 16 µL of each cell lysate were analyzed by HTRF Phospho SLP-76(Ser376) assays.

Kinetics of cell stimulation

This assay was carried out with a two plate protocol. 400,000 Jurkat cells per well were plated in a 96-well culture plate at 25µL/well in complete RPMI culture medium with 10% FBS. Cell stimulation was performed using anti CD3 Ab at the final concentration of 20µg/mL for different stimulation times (min). After stimulation, cells were lysed with 10 µL of 4X supplemented lysis buffer. 16 µL of each cell lysate for the different conditions were analyzed by HTRF Phospho SLP-76(Ser376) and Total SLP-76.

Monitoring of the immune checkpoint inhibitor PD-L1 activity

As Phospho SLP-76 (Tyr376) is a readout of T cell activation, it was used to monitor the inhibitory effect of PD-L1 recombinant protein on T cell activation. This assay was performed using the two-plate assay protocol. 400 000 Jurkat cells were plated in 25 µL of complete RPMI culture medium with 10% FBS. Cells were pre-incubated 24 hours with different concentrations of PD-L1 recombinant protein. Cells were then stimulated for 2.5 min with 20 µg / mL anti-CD3 Ab (final concentration) and lysed directly with10 µL of 4X supplemented lysis buffer, then incubated 30 min at RT. 16µL of each sample were analyzed using Phospho SLP-76 (Ser376) and Total SLP-76 assays. Phospho SLP-76 results were normalized with the Total SLP-76 amount and plotted on a graph.

A slight but significant inhibitory effect of PD-L1 recombinant protein was recorded with the Phospho SLP-76 (Ser376) assay at the final concentration of 40 µg/mL

Monitoring of the immune checkpoint inhibitor PD-L1 activity

Function and regulation of SLP-76

TCR engagement promotes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytosolic side of the TCR/CD3 complex by lymphocyte protein tyrosine kinase (Lck). Zap-70 is recruited to the TCR/CD3 complex, where it becomes phosphorylated and activated. ZAP-70 phosphorylates SLP-76. Activated SLP-76 translocates to the plasma membrane and promotes a multi-protein complex, which participates in the activation, survival, and proliferation of T-Lymphocytes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

PI3K/AKT/mTor translational control pathway - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

TCR signaling investigation with HTRF phospho assays

Study a pathway of interest in PBMC and T cells - Application Notes

From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery

One technology across all samples - Application Notes

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

Multi-tissue cellular modeling and anlysis of insulin signaling - Posters

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

A single technology for 2D cells, 3D cells, and xenograft models - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening

In collaboration with Bayer - Scientific Presentations

Product Insert SLP76 P-S376 Kit / 63ADK076PET

63ADK076PET - Product Insert

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

Product Insert SLP76 P-S376 Kit / 63ADK076PEG-63ADK076PEH

63ADK076PEG-63ADK076PEH - Product Insert

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Webinar: What's next for CAR T-cells immunotherapy

Featuring a panel of experts - Videos

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Innovative solutions in Virology research

Kits and reagents for virology research - Flyers

Assays for neurosciences research

Advance your research on neurodegenerative diseases - Flyers

Advance your research on Fibrosis

Kits and reagents for Fibrosis research - Flyers

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Safety Data Sheet (DEU) SLP76 P-S376 Kit / 63ADK076PEG

63ADK076PEG - Safety Data Sheet

Safety Data Sheet (ELL) SLP76 P-S376 Kit / 63ADK076PEG

63ADK076PEG - Safety Data Sheet

Safety Data Sheet (FRA-FR) SLP76 P-S376 Kit / 63ADK076PEG

63ADK076PEG - Safety Data Sheet

Safety Data Sheet (ITA) SLP76 P-S376 Kit / 63ADK076PEG

63ADK076PEG - Safety Data Sheet

Safety Data Sheet (SPA) SLP76 P-S376 Kit / 63ADK076PEG

63ADK076PEG - Safety Data Sheet

Safety Data Sheet (ENG-GB) SLP76 P-S376 Kit / 63ADK076PEG

63ADK076PEG - Safety Data Sheet

Safety Data Sheet (ENG-US) SLP76 P-S376 Kit / 63ADK076PEG

63ADK076PEG - Safety Data Sheet

Safety Data Sheet (DEU) SLP76 P-S376 Kit / 63ADK076PEH

63ADK076PEH - Safety Data Sheet

Safety Data Sheet (ELL) SLP76 P-S376 Kit / 63ADK076PEH

63ADK076PEH - Safety Data Sheet

Safety Data Sheet (FRA-FR) SLP76 P-S376 Kit / 63ADK076PEH

63ADK076PEH - Safety Data Sheet

Safety Data Sheet (ITA) SLP76 P-S376 Kit / 63ADK076PEH

63ADK076PEH - Safety Data Sheet

Safety Data Sheet (SPA) SLP76 P-S376 Kit / 63ADK076PEH

63ADK076PEH - Safety Data Sheet

Safety Data Sheet (ENG-GB) SLP76 P-S376 Kit / 63ADK076PEH

63ADK076PEH - Safety Data Sheet

Safety Data Sheet (ENG-US) SLP76 P-S376 Kit / 63ADK076PEH

63ADK076PEH - Safety Data Sheet

Safety Data Sheet (DEU) SLP76 P-S376 Kit / 63ADK076PET