Phospho-SLP-76 (Ser376) & total SLP-76 Cellular Assay Kits

Phospho- and total SLP-76 assays for monitoring T lymphocyte activation

SLP-76 is a cytoplasmic adapter protein, expressed in multiple hematopoietic lineages.  Phospho-SLP-76 creates a scaffold on which key signaling complexes are built, and plays a critical role in T-cell development and activation. Following TCR engagement, SLP-76 is phosphorylated by Zap-70 and translocates to the plasma membrane where it promotes the assembly of a multi protein complex. This complex participates in the activation, survival, and proliferation of T-Lymphocytes.

Cisbio's cell-based homogeneous HTRF® phospho-SLP-76 (Ser376) immunoassay enables the quantitative detection of SLP-76 phosphorylated on Serine 376. The HTRF® total SLP-76 assay kit is designed for the quantitative detection of total SLP-76, phosphorylated and unphosphorylated, to normalize the phosphorylation status of SLP-76 with respect to its steady-state level in cells.

The buffers of both HTRF® phospho- and total SLP-76 assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total SLP-76 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-SLP76 antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with SLP-76, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.
The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total SLP-76 can be quantitatively detected using the HTRF phospho-SLP-76 (Ser376) and total SLP-76 cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are first plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

Product performances

1. HTRF assay compared to Western Blot using phospho & 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.

2. 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.

3. 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.

4. Monitoring of the immune checkpoint inhibitor PD-L1 activity

As Phospho SLP-76 (Ser376) is a readout of T cell activation, we used it to monitor the inhibitory effect of PDL-1 recombinant protein on T cell activation.
This assay was carried out with a two plate protocol.
400,000 Jurkat cells per well were plated in a 96-well culture plate in 25µL of complete RPMI culture medium with 10% FBS. The cells were then pre-incubated for 24 hours with different concentrations of PDL-1 recombinant protein. After incubation, the Jurkat cells were stimulated using anti-CD3 Ab at 20 µg/mL (final concentration) for 2.5 min and cell lysis was directly done by adding 10 µL of 4X supplemented lysis buffer, followed by incubation at RT 30 min. 16µL of lysate for each condition were analyzed using Phospho SLP-76 and Total SLP76 assays.
Each Phospho SLP-76 result was next 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.

Simplified pathway

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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
SLP-76 phospho S376 kit - 500 tests63ADK076PEG
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SLP-76 phospho S376 kit - 10,000 tests63ADK076PEH
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SLP-76 phospho S376 kit - 1 x 96 tests63ADK076PET
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SLP-76 total kit - 500 tests63ADK077PEG
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SLP-76 total kit - 10,000 tests63ADK077PEH
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Companion products

DescriptionCat. noProduct insertMSDS
SLP-76 phospho S376 control lysate63ADK076TDA
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SLP-76 total control lysate63ADK077TDA
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Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
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Literature