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

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Rapid-Act T Cell Activation Kit (Mouse, Anti-CD3/CD28): Image 1
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.
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To Purchase # 86772
Cat. # Size Qty. Price
86772S		 1 Kit  (50 tests)
$ 357

Custom Formulation

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

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#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.

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