Testing Data
(Intracellular Staining of PBMC. 0.5 ug of antibody was used. Red: Human TLR3 PE. Green: Isotype control (mouse IgG1PE) was used. Intracellular FLOW kit was used.)
FLOW IntraCellular Staining Kit
FLOW IntraCellular Staining Kit (IC FLOW)
Applications
Flow Cytometry, Functional Assay
Synonyms
FLOW IntraCellular; FLOW IntraCellular Staining Kit (IC FLOW); FLOW IntraCellular staining kit
Applicable Applications for FLOW IntraCellular staining kit
Flow Cytometry (FC/FACS)
Application Notes
Protocol: 1. Determine number of cells required for staining. Each sample contains 0.5 -1 x 10^6 cells in 50ul of media or staining buffer. The following controls are needed for the experiment. Unstained cells (no antibodies were added), cells with isotype control and cells with secondary antibody (if secondary antibody was used). 2. Cetrifuge cells at 1000 RPM for 10 minutes and decant supernatant. 3. Resuspend cells with appropriate volume of Fixation buffer. Incubate in ice for for 30 minutes. 4. Centrifuge cells 1000 RPM for 10 minutes and decant supernatant. 5. Resuspend pellet with appropriate volume of 1X permeabilization buffer. 6. Aliquote 1x10^6 cells in 50 ul to the desired number of flow tubes. Dilute primary antibodies in 50 ul of 1X permeabilization buffer. Add diluted antibodies into 50 ul of cells. Mix antibodies in cells suspension thoroughly. 7. Incubate in ice for 30 minutes. Wash cells in 2-3 ml of 1X permeabilization buffer. Centrifuge 1000 RPM for 10 minutes. Decant supernatant carefully. If primary antibody is fluorescent-labeled, resuspent pellet in 300-400 ul of staining buffer, analyze samples. Protected from light. If primary antibody is not labeled, proceed with step 8. 8. Dilute secondary antibody (PE, FITC, APC or biotin labeled) in 50 ul of permeabilization buffer per sample. 9. Resuspend cells in diluted secondary antibody. Incubate in ice for 30 minutes. (protected from light). 10. Wash cells in 3-4 ml of permeabilization buffer. Cetrifuge at 1000 RPM for 10 minutes. Decant supernatant. 11. After decanting, add 300-400ul of Staining Buffer to each tube. If not analyzing same day, samples can be stored over night in dark at 4 degrees C. Samples can be analyzed in Flow Cytometer according to the manufacturer protocol.
Kit Components
Fixation Buffer (1X) - 60 ml, Permeabilization Buffer (10X)- 2 X 60 ml, Staining Buffer - (1X) - 60 ml
Preparation and Storage
Store at 2-8 degree C
Testing Data
(Intracellular Staining of PBMC. 0.5 ug of antibody was used. Red: Human TLR3 PE. Green: Isotype control (mouse IgG1PE) was used. Intracellular FLOW kit was used.)
Testing Data
(Intracellular Staining of PBMC. 0.5 ug of antibody was used. Red: Human TLR3 PE. Green: Isotype control (mouse IgG1PE) was used. Intracellular FLOW kit was used.)
Related Product Information for FLOW IntraCellular staining kit
The Intracellular FLOW Staining Kit allows detection antibodies to access the intracellular protein in Flowcytometry analysis. This kit is specially formulated to reduce non-specific staining of fluorochrome-labelled antibodies and to increase fluorescence signaling. First, live cells can be fixed with the Fixation buffer, which cross-links the protein. Second, the 1X Permeabilization Buffer creates holes in the membrane, allowing the antibodies enter into the cells effectively. Washing steps and antibody dilutions are done using 1X Permeabilization Buffer. The final cell resuspension prior to analysis is done using The Staining Buffer. Permeabilization Buffer is supplied as a 10X solution. Prior to use, this should be diluted 10 fold in distilled water. (1ml of Permeabilization Buffer with 9 ml of distilled water). 10X Permeabilization buffer has natural tendency to precipitate, however, its function is not affected. 1X solution can be filtered.
Product Categories/Family for FLOW IntraCellular staining kit
Similar Products
Product Notes
The FLOW IntraCellular (Catalog #AAA668927) is a Staining Kit and is intended for research purposes only. The product is available for immediate purchase. AAA Biotech's FLOW IntraCellular can be used in a range of immunoassay formats including, but not limited to, Flow Cytometry (FC/FACS). Protocol: 1. Determine number of cells required for staining. Each sample contains 0.5 -1 x 10^6 cells in 50ul of media or staining buffer. The following controls are needed for the experiment. Unstained cells (no antibodies were added), cells with isotype control and cells with secondary antibody (if secondary antibody was used). 2. Cetrifuge cells at 1000 RPM for 10 minutes and decant supernatant. 3. Resuspend cells with appropriate volume of Fixation buffer. Incubate in ice for for 30 minutes. 4. Centrifuge cells 1000 RPM for 10 minutes and decant supernatant. 5. Resuspend pellet with appropriate volume of 1X permeabilization buffer. 6. Aliquote 1x10^6 cells in 50 ul to the desired number of flow tubes. Dilute primary antibodies in 50 ul of 1X permeabilization buffer. Add diluted antibodies into 50 ul of cells. Mix antibodies in cells suspension thoroughly. 7. Incubate in ice for 30 minutes. Wash cells in 2-3 ml of 1X permeabilization buffer. Centrifuge 1000 RPM for 10 minutes. Decant supernatant carefully. If primary antibody is fluorescent-labeled, resuspent pellet in 300-400 ul of staining buffer, analyze samples. Protected from light. If primary antibody is not labeled, proceed with step 8. 8. Dilute secondary antibody (PE, FITC, APC or biotin labeled) in 50 ul of permeabilization buffer per sample. 9. Resuspend cells in diluted secondary antibody. Incubate in ice for 30 minutes. (protected from light). 10. Wash cells in 3-4 ml of permeabilization buffer. Cetrifuge at 1000 RPM for 10 minutes. Decant supernatant. 11. After decanting, add 300-400ul of Staining Buffer to each tube. If not analyzing same day, samples can be stored over night in dark at 4 degrees C. Samples can be analyzed in Flow Cytometer according to the manufacturer protocol. Researchers should empirically determine the suitability of the FLOW IntraCellular for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "FLOW IntraCellular, Staining 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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