Cell Stimulation and Protein Transport Inhibitor Kit (AKES084)
The Assay Genie Cell Stimulation and Protein Transport Inhibitor Kit is an optimized, broad-spectrum immune cell stimulator and inhibitor that induces a variety of cells in vitro to produce cytokines while blocking the transport of secreted proteins to the extracellular space. The kit combines a Cell Stimulation MIX of Phorbol 12-Myristate 13-Acetate (PMA) and Ionomycin, which drives cell activation and cytokine secretion, with a Protein Transport Inhibitor MIX of Monensin and Brefeldin A, which retains cytokines inside the cell for detection after fixation and permeabilization. It is suitable for measuring cytokine content or activity in cell culture supernatants by ELISA and for intracellular cytokine staining of single cell suspensions such as mouse spleen and human peripheral blood T lymphocytes.
Product Name:
Cell Stimulation and Protein Transport Inhibitor Kit
SKU:
AKES084
Size:
50 Assays
Detection Principle:
Cell Stimulation & Protein Transport Inhibition
Form:
Powder (with solvent)
Applications:
Cytokine content or activity detection in cell culture supernatant; intracellular factor detection
Sample Type:
Single cell suspensions (e.g. mouse spleen T lymphocytes, human peripheral blood T lymphocytes)
Shipping:
Gel Pack
Usage:
For Research Use Only
No
Component Name
50 Assays
Storage
1
Cell Stimulation MIX Powder (50 µg)
50 µg × 1 vial
-20°C/-80°C (protected from light)
2
Cell Stimulation MIX Solvent
120 µL
-20°C (protected from light)
3
Protein Transport Inhibitor MIX Powder (200 µg)
200 µg × 1 vial
-20°C/-80°C (protected from light)
4
Technical Manual
One copy
Note: Do not mix reagents from different kits. For reagents provided in small volumes, centrifuge briefly before use to ensure complete recovery of the contents.
Reagent
Preparation
500× Cell Stimulation MIX
Add 100 µL Cell Stimulation MIX Solvent to one vial of Cell Stimulation MIX Powder (50 µg) and mix fully.
1000× Protein Transport Inhibitor MIX
Add 50 µL 33% DMSO solution (self-prepared) to one vial of Protein Transport Inhibitor MIX Powder (200 µg) and mix fully.
33% DMSO Solution
Mix 670 µL sterile ultrapure or deionized water with 330 µL anhydrous DMSO. Store at -20°C away from light.
Note: Centrifuge the powder at 8,000–10,000 × g for 1 minute so that it collects at the bottom of the tube before reconstitution.
Step
Application 1: Cytokine Content or Activity Detection in Cell Culture Supernatant
1
Prepare a single cell suspension in complete medium and adjust the cell density to 1–2×106/mL.
Note: Keep cell density below 2×106/mL, as high density reduces activation efficiency. Ensure cells are in good condition before stimulation, especially freshly prepared primary cells.
2
Add 2 µL of 500× Cell Stimulation MIX per 1 mL of cell suspension and incubate at 37°C, 5% CO2 for 4–18 hours. Determine the optimal induction time for each cytokine with a pilot experiment (see Reference Induction Conditions).
3
Collect the cell culture supernatant for immediate detection of cytokine content or activity (e.g. by ELISA), or store at -80°C for later use.
Step
Application 2: Intracellular Factor Detection
1
Prepare a single cell suspension in complete medium and adjust the cell density to 1–2×106/mL.
Note: Keep cell density below 2×106/mL, as high density reduces activation efficiency. Ensure cells are in good condition before stimulation, especially freshly prepared primary cells.
2
Add 2 µL of 500× Cell Stimulation MIX per 1 mL of cell suspension and incubate at 37°C, 5% CO2 for 1–1.5 hours.
3
Add 1 µL of 1000× Protein Transport Inhibitor MIX per 1 mL of cell suspension and incubate at 37°C, 5% CO2 for 5–16 hours. Determine the optimal induction time for each cytokine with a pilot experiment (see Reference Induction Conditions).
4
Collect the cell suspension, centrifuge at 200–300 × g for 5 minutes and discard the supernatant. The cell pellet can be used for intracellular factor detection after fixation.
