View Featured Offers >>
86772
Rapid-Act T Cell Activation Kit (Mouse, Anti-CD3/CD28)
Cellular Assay Kits
Assay Kit

Rapid-Act T Cell Activation Kit (Mouse, Anti-CD3/CD28) #86772

Citations (0)
Flow cytometric analysis of mouse splenocytes, untreated (left column) or treated with Rapid-Act T Cell Activation Kit (Mouse, Anti-CD3/CD28) (15 min; right column), using Phospho-SLP-76 (Ser376) (E3G9U) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #47876 (top row) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (bottom row), and co-stained with CD3 (17A2) Rat mAb (APC Conjugate) #24265.
To Purchase # 86772
Cat. # Size Qty. Price
86772S
1 Kit  (50 tests)

Product Includes Quantity (with Count) Storage Temp
Rapid-Act Anti-mouse CD3 Hamster mAb 1 x 125 µg +4°C
Rapid-Act Anti-mouse CD28 Mouse mAb 1 x 125 µg +4°C
Rapid-Act Synthetic Immune Synapse 1 x 500 µl +4°C

Storage

Store at 4ºC. All components in this kit are stable for 12 months when kept in the original format and stored at 4ºC. Once reconstituted, store the Anti-mouse CD3 and Anti-mouse CD28 at -80ºC. Aliquot to avoid multiple freeze/thaw cycles.

Protocol

PRINT

View >Collapse >

#86772 Rapid-Act T Cell Activation Kit (Mouse, Anti-CD3/CD28)

A. Solutions and Reagents

Supplied Reagents

  1. Rapid-Act Anti-mouse CD3 Hamster mAb, lyophilized, 125 µg (#46345)
  2. Rapid-Act Anti-mouse CD28 Mouse mAb, lyophilized, 125 µg (#63606)
  3. Rapid-Act Synthetic Immune Synapse, 500 µl (#68999)

Additional Reagents (Not Supplied)

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml reverse osmosis deionized (RODI) or equivalent grade water
  2. Cell Culture Medium (assay dependent)

B. Reagent Preparation

NOTE: Volumes are suitable for stimulation and expansion of 1 x 106 cells. For different cell numbers, adjust volumes according to Table 1.

NOTE: We recommend preparing the Rapid-Act CD3/CD28 complex fresh for each experiment. If necessary, store pre-mixed components at 4 °C. Do not freeze.

  1. Dissolve each Rapid-Act antibody in 500 µl 1X PBS. Store Rapid-Act antibody suspensions in aliquots at -80 °C.
  2. In a microcentrifuge tube, combine 10 µl Rapid-Act Anti-mouse CD3 with 10 µl Rapid-Act Anti-mouse CD28. Mix well. Add 10 µl of Rapid-Act Synthetic Immune Synapse and mix well. Incubate the mixture for at least 20 min at 4 °C to generate Rapid-Act CD3/CD28 complex.

C. Cell Preparation

  1. Collect cells of interest and pellet by centrifugation. Splenocytes, enriched T cells, or cell lines may be used. Recommended cell numbers and volumes in every step are identical to pre-isolated T cells.
  2. Resuspend cells in cell culture medium at a concentration of 0.2 x 106 – 1 x 106 cells per 1 ml (optimal conditions should be titrated).

Table 1: Recommended volumes for different cell numbers

96-well 48-well 24-well
Cell number 5 – 8 x 104 2 – 5 x 105 0.5 – 1 x 106
Cell culture medium (ml) 0.1 – 0.2 0.5 – 1.0 1.0 – 2.0
Rapid-Act CD3/CD28 complex (µl) 3 15 30

D. T cell Activation and Expansion Procedure

1. T cell activation

  1. Seed 0.5 – 1 x 106 cells in 1 – 2 ml cell culture medium (or use volumes and cell densities as determined above).
  2. Add 30 µl Rapid-Act CD3/CD28 complex to the cells and mix gently. Incubate cell suspension in a humidified CO2 incubator at 37 °C, according to your experimental setup.
  3. Harvest activated T cells, wash twice with excess 1X PBS by centrifugation, and use directly for further analysis.

2. T cell expansion

NOTE: Activation markers CD25 and CD69 should be upregulated after 48 h.

  1. Activate T cells following steps 1 and 2 in T cell activation section above.
  2. Examine culture daily, observing cell size, shape, and cluster formation using a microscope. Count the cells at least every 2-3 days to evaluate cell density; do not exceed 2.5 x 106 cells/ml. If the cell medium turns yellow or cell density is too high, split cultures back to a density of 0.2 – 1 x 106 cells/ml into a new container of appropriate size.
  3. Restimulation of the cells might be necessary after 2 – 3 days in culture, with signs of exhaustion typical after 7 – 10 days. Repeat addition of Rapid-Act CD3/CD28 complex as described in T cell activation to restimulate cells.
  4. Harvest expanded T cells, wash twice with excess 1X PBS by centrifugation, and use directly for further analysis.

Protocol Id: 2845

Product Description

The Rapid-Act T Cell Activation Kit (Mouse, Anti-CD3/CD28) provides the necessary reagents for easy, single-step induction of mouse T cell activation and proliferation. The included Synthetic Immune Synapse particles pre-cluster the CD3 and CD28 antibodies for thorough T cell activation, and are small enough to minimize cell clumping. Because the reagents are added to the cells in a single step, the kit enables accurate time course studies for short-term T cell stimulation. The kit can also be used for longer-term T cell proliferation and exhaustion assays. No modification or removal is necessary prior to subsequent analysis by flow cytometry.

Specificity / Sensitivity

Species Reactivity:

Mouse

Background

T cells are activated by signaling through ITAM (immunoreceptor tyrosine-based activation motif)-containing CD3 signaling chains that associate with the T cell receptor (1). Co-stimulation through CD28 on T cells provides an additional signal required for effective T cell activation (2). Antibody-based binding and aggregation of CD3 and CD28 proteins simulates the engagement of a T cell with an antigen presenting cell, and results in T cell activation.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit our Trademark Information page.