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
1.
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.
2.
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.
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 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.
|
Cautions
1.
This kit is for research use only.
2.
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.
3.
Please take safety precautions and
follow the procedures of laboratory reagent operation.
4.
Please store the product at the
appropriate temperature to avoid failure.
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