Total Price: $0.00
Contact Information
Protein Transport Inhibitor MIX
SKU: E-CK-A013-500
To better serve you, we would like to discuss your specific requirement.
Please Contact Us for a quote.
Protein Transport Inhibitor MIX
Introduction
Elabscience® Protein Transport Inhibitor MIX is mainly composed of Monensin and Brefeldin A, which can be used in combination with Cell Stimulation MIX [E-CK-A019] 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.
Reagent Preparation
1000×Protein Transport Inhibitor MIX: Add 50 μL absolute ethanol (self-prepared) to a vial of Protein Transport Inhibitor MIX and mix fully.
Note: Centrifuge at 2000~10000×g for several seconds before use and then open the cover for use. Absolute ethanol is volatile, please keep it sealed properly.
Experimental Procedure
-
Prepare the single cell suspension with complete medium (self-prepared), and adjust the cell density to 1~2×106/mL.
Note: The cell density should not be too high, and the maximum density should be less than 2×106/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. - Add 2 μL of 500× Cell Stimulation MIX [E-CK-A019] to each 1mL of cell suspension, and incubate the cells at 37°C, 5%CO2 for 1.5~1 h.
- 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).
- Collect cell suspension, centrifuge at 200~300×g for 5 min, discard the supernatant and collect the cell pellet which could be used for subsequent intracellular factor detection after fixation.
Table 1: Reference of inducing condition of intracellular factors
|
Species |
Target cell |
Cytokines/chemokines |
Induction time |
|
Mouse |
Spleen T lymphocytes |
IL-17A |
5~6 h |
|
IFN-γ |
5~6 h |
||
|
IL-4 |
5~6 h |
||
|
IL-2 |
5~6 h |
||
|
IL-10 |
5~6 h |
||
|
IL-6 |
5~6 h |
||
|
Human |
Peripheral blood T lymphocytes |
IL-17A |
5~6 h |
|
IFN-γ |
5~6 h |
||
|
IL-4 |
5~6 h |
||
|
IL-2 |
5~6 h |
||
|
IL-6 |
5~6 h |
||
|
IL-10 |
5~6 h |
||
|
IL-21 |
5~6 h |
Troubleshooting
|
Symptoms |
Causes |
Comments |
|
Cytokines were detected in supernatants but not in cells |
The incubation time of 1000×Protein Transport Inhibitor MIX is insufficient. |
Appropriately increase the incubation time of 1000×Protein Transport Inhibitor MIX. |
|
More cell loss |
Centrifugal conditions are not appropriate. |
Unfixed living cells centrifugal force is less than 300×g, the speed of acceleration is less than 3,the speed of deceleration is less than 2, which can greatly reduce the cell loss caused by centrifugation. |
|
Too many cells, inadequate fixation. |
Increase fixed liquid volume and extend fixation time. |
Storage
- 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.
Notes
- This kit is for research use only.
- 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 factors to be secreted outside the cell.
- Please take safety precautions and follow the procedures of laboratory reagent operation.
- Please store the product at the appropriate temperature to avoid failure.