Protein Transport Inhibitor MIX Power

K083-3

50T/200T

Powder reagents can be stored for 1 year in the dark at -20°C and 2 years in the dark at-80°C.

The dissolved powder can be stored at-20°C for 6 months, or stored at-80°C for 1 year after subpackaged.

Protein Transport Inhibitor MIX is mainly composed of Monensin and Brefeldin A, which can be used in combination with Cell Stimulation MIX to prevent the loss of cytokine transport.

After cell membrane rupture, cytokines can be detected. It can also be used alone to block protein transport from the Golgi apparatus and endoplasmic reticulum in cells.

1000×Protein Transport Inhibitor MIX: Add 50 μL 33% DMSO solution (self-prepared) to a vial of Protein Transport Inhibitor MIX Powder ( 200 μg ) and mix fully.

Note: Please centrifuge the powder at 8000~10000×g for 1 min, so that the powder will be gathered at the bottom of the tube before reagent preparation;

33% DMSO solution can be prepared by mixing 670 μL of sterile ultrapure water or sterile deionized water with 330 μL of anhydrous DMSO, then stored at-20 °C away from light.

1.Prepare the single cell suspension with complete medium (self-prepared), and adjust the cell density to 1~2×10^6/mL.

Note: The cell density should not be too high, and the maximum density should be less than 2×10^6/mL, high cell density will affect cell activation efficiency. Make sure the cells are in good condition before stimulation, especially for freshly prepared primary cells.

2.Sample preparation Add 2 μL of 500× Cell Stimulation MIX to each 1mL of cell suspension, and incubate the cells at 37°C, 5%CO2 for 1.5~1 h.

3.Add 1 μL of 1000×Protein Transport Inhibitor MIX to each 1mL of cell suspension, and incubate the cells at 37°C, 5%CO2 for 5~16 h (It is recommended to determine the optimal induction time by setting up a pre-experiment with different induction times for the cytokines to be tested. The common induction time can be refer to table 1).

4.Collect cell suspension, centrifuge at 200~300×g for 5min, discard the supernatant and collect the cell pellet which could be used for subsequent intracellular factor detection after fixation.

1.This assay kit is for Research Use Only. We will not response for any arising problems or legal responsibilities causing by using the kit for clinical diagnosis or other purpose.

2.Please take safety precautions and follow the procedures of laboratory reagent operation.

3.Please store the product at the appropriate temperature to avoid failure.

4. Due to the effect of Brefeldin A in Protein Transport Inhibitor MIX on CD69, it is recommended not to add Protein Transport Inhibitor MIX when detecting CD69. However, this operation may cause Intracellular factor to be secreted outside the cell.