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Total SLP-76 cellular kit HTRF®

The Total SLP-76 kit detects cellular SLP-76 and can be used as a normalization assay with our phospho-SLP-76 kit to assist in optimal monitoring of T-lymphocyte activation.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The Total SLP-76 kit detects cellular SLP-76 and can be used as a normalization assay with our phospho-SLP-76 kit to assist in optimal monitoring of T-lymphocyte activation.
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Overview

The total SLP-76 cellular assay kit is designed to monitor the expression level of SLP-76, whether phosphorylated or unphosphorylated. It is compatible with our Phospho-SLP-76 (Ser376) kit, and enables the analysis of phosphorylated and total proteins from a single sample for better readout of T-lymphocyte activation.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-SLP-76 assay principle

The Total-SLP-76 assay quantifies the expression level of SLP-76 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-SLP-76 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of SLP-76 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total SLP-76 assay principle

Total-SLP-76 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 Total SLP-76 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total SLP-76 2-plate assay protocol

Total-SLP-76 1-plate assay protocol

Detection of total SLP76 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.
Total SLP-76 1-plate assay protocol

HTRF assay compared to Western Blot using total SLP-76 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.
HTRF assay compared to Western Blot using total SLP-76 cellular assays on human Jurkat cells

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.
Kinetics of cell stimulation

Monitoring of the immune checkpoint inhibitor PD-L1 activity

As Phospho SLP76 (Ser396) 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
Pathway of SLP-76 cell signaling

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

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Open R&D: Sanofi Access Platform

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Lysis buffer compatibility

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Species compatibility

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Product Insert SLP76 total Kit / 63ADK077PEG-63ADK077PEH

63ADK077PEG-63ADK077PEH - Product Insert

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Plate Reader Requirement

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