Application Data
Cell stimulation and Protein Transport Inhibitor Kit
Cell stimulation and Protein Transport Inhibitor Kit
Synonyms
Cell stimulation and Protein Transport Inhibitor; N/A; Cell stimulation and Protein Transport Inhibitor Kit; Cell stimulation and Protein Transport Inhibitor kit
Reagent Preparation
1) 500×Cell Stimulation MIX
Add 100 uL Cell Stimulation MIX Solvent to dissolve a vial of Cell Stimulation MIX and mix fully.
2) 1000×Protein Transport Inhibitor MIX
Add 50 uL 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.
Add 100 uL Cell Stimulation MIX Solvent to dissolve a vial of Cell Stimulation MIX and mix fully.
2) 1000×Protein Transport Inhibitor MIX
Add 50 uL 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
Application 1: Cytokine content or activity detection in cell culture supernatant
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 uL of 500× Cell Stimulation MIX to each 1 mL of cell suspension, and incubate the cells at 37 degree C, 5%CO2 for 4~18 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).
3. Collect cell culture supernatant for the subsequent detection or store at-80 degree C for later use (the supernatant contains a variety of cytokines secreted by cells, which can be used to detect the content and activity of cytokines by ELISA or other biochemical reagents).
Application 2: Intracellular factor detection
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 uL of 500× Cell Stimulation MIX to each 1mL of cell suspension, and incubate the cells at 37 degree C, 5%CO2 for 1.5~1 h.
3. Add 1 uL of 1000×Protein Transport Inhibitor MIX to each 1mL of cell suspension, and incubate the cells at 37 degree 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.
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 uL of 500× Cell Stimulation MIX to each 1 mL of cell suspension, and incubate the cells at 37 degree C, 5%CO2 for 4~18 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).
3. Collect cell culture supernatant for the subsequent detection or store at-80 degree C for later use (the supernatant contains a variety of cytokines secreted by cells, which can be used to detect the content and activity of cytokines by ELISA or other biochemical reagents).
Application 2: Intracellular factor detection
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 uL of 500× Cell Stimulation MIX to each 1mL of cell suspension, and incubate the cells at 37 degree C, 5%CO2 for 1.5~1 h.
3. Add 1 uL of 1000×Protein Transport Inhibitor MIX to each 1mL of cell suspension, and incubate the cells at 37 degree 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.
Preparation and Storage
Store at -20 degree C, shading light
Estimated Expiration: 12 months
Shipping: Ice bag
Estimated Expiration: 12 months
Shipping: Ice bag
Application Data
Application Data
Application Data
Application Data
Related Product Information for Cell stimulation and Protein Transport Inhibitor kit
Background: Cell Stimulation and Protein Transport Inhibitor Kit is an optimized broad-spectrum immune cell stimulator and inhibitor that can induce and stimulate a variety of cells in vitro to produce cytokines and block the transport of secreted proteins to the extracellular. Cell stimulation and Protein Transport Inhibitor Kit is mainly composed of Cell Stimulation MIX and Protein Transport Inhibitor MIX. Cell Stimulation MIX is a mixture of Phorbol 12-Myristate 13-Acetate (PMA) and Ionomycin, which can induce various cell activation and secrete cytokines. Protein Transport Inhibitor MIX is mainly composed of Monensin and Brefeldin A, which can prevent the loss of cytokine transport. After cell membrane rupture, cytokines can be detected.
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Product Notes
The Cell stimulation and Protein Transport Inhibitor (Catalog #AAA178129) is a Kit and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "Cell stimulation and Protein Transport Inhibitor, Kit" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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