Intracellular Fixation/Permeabilization Buffer Kit
Intracellular Fixation/Permeabilization Buffer Kit
Synonyms
Intracellular Fixation/Permeabilization Buffer; N/A; Intracellular Fixation/Permeabilization Buffer Kit; Intracellular Fixation/Permeabilization Buffer kit
Instruction
Dilute Permeabilization Buffer (5×) with deionized water to 1×Permeabilization Working Solution before use. For example, take 1 mL Permeabilization Buffer (5×) to 4 mL deionized water and mix fully to prepare 1× Permeabilization Working Solution.
Experimental Procedure
1. Take 1×10^6 cells in 100 uL suspension into the tube per sample.
2. [Optional] Stain cells with a Fixable Viability Dye (self-prepared).
3. [Optional] Block Fc receptors in cell suspensions according to experimental requirements.
4. Stain cell surface markers as need.
5. After incubating with the cell surface marker, add 2 mL of PBS (with 1% BSA, self-prepared) or Cell Staining Buffer [MBS2567126], centrifuge at 300×g for 5 min, discard the supernatant.
6. Resuspend the cells with 200 uL of PBS (with 1% BSA, self-prepared) or Cell Staining Buffer [MBS2567126]. Then add 200uL of Fixation Buffer, incubate the cells at room temperature for 30~60 min in the dark (please extend the incubation time to 60 min when the room temperature is lower than 25 degree C).
7. Add 1 mL of 1×Permeabilization Working Solution to each tube and mix fully, centrifuge at 600×g for 5 min and discard the supernatant.
8. Resuspend the cells with 100 uL of 1×Permeabilization Working Solution. Add the appropriate volume of intracellular antibody or corresponding isotype control and incubate at least 30 min at room temperature in the dark.
9. Add 2 mL of PBS (with 1% BSA) or Cell Staining Buffer [MBS2567126] to each tube and centrifuge at 600×g for 5 min, discard the supernatant.
10. Resuspend the cells with appropriate PBS (with 1% BSA) or Cell Staining Buffer [MBS2567126], then analyze the samples by flow cytometry.
2. [Optional] Stain cells with a Fixable Viability Dye (self-prepared).
3. [Optional] Block Fc receptors in cell suspensions according to experimental requirements.
4. Stain cell surface markers as need.
5. After incubating with the cell surface marker, add 2 mL of PBS (with 1% BSA, self-prepared) or Cell Staining Buffer [MBS2567126], centrifuge at 300×g for 5 min, discard the supernatant.
6. Resuspend the cells with 200 uL of PBS (with 1% BSA, self-prepared) or Cell Staining Buffer [MBS2567126]. Then add 200uL of Fixation Buffer, incubate the cells at room temperature for 30~60 min in the dark (please extend the incubation time to 60 min when the room temperature is lower than 25 degree C).
7. Add 1 mL of 1×Permeabilization Working Solution to each tube and mix fully, centrifuge at 600×g for 5 min and discard the supernatant.
8. Resuspend the cells with 100 uL of 1×Permeabilization Working Solution. Add the appropriate volume of intracellular antibody or corresponding isotype control and incubate at least 30 min at room temperature in the dark.
9. Add 2 mL of PBS (with 1% BSA) or Cell Staining Buffer [MBS2567126] to each tube and centrifuge at 600×g for 5 min, discard the supernatant.
10. Resuspend the cells with appropriate PBS (with 1% BSA) or Cell Staining Buffer [MBS2567126], then analyze the samples by flow cytometry.
Cautions
1. This kit is for research use only.
2. Permeabilization Buffer (5×) may precipitate, and it will not affect the use effect.
3. For samples with red blood cells, please lyse red blood cells first.
4. The fixation and permeabilization steps may alter the light scatter properties of cells and may increase non-specific background staining. The addition of BSA or fetal calf serum (FBS) in the staining buffer help to reduce non-specific background. It is recommended to use Fixable Viability Dye to eliminate the interference of dead cells in the data analysis process.
5. This product is compatible with most commercially available flow antibodies, but some antigenic determinants are sensitive to fixatives and the fixation time needs to be optimized for the situation.
6. For your safety and health, please wear the lab coat and disposable gloves before the experiments.
2. Permeabilization Buffer (5×) may precipitate, and it will not affect the use effect.
3. For samples with red blood cells, please lyse red blood cells first.
4. The fixation and permeabilization steps may alter the light scatter properties of cells and may increase non-specific background staining. The addition of BSA or fetal calf serum (FBS) in the staining buffer help to reduce non-specific background. It is recommended to use Fixable Viability Dye to eliminate the interference of dead cells in the data analysis process.
5. This product is compatible with most commercially available flow antibodies, but some antigenic determinants are sensitive to fixatives and the fixation time needs to be optimized for the situation.
6. For your safety and health, please wear the lab coat and disposable gloves before the experiments.
Preparation and Storage
Store at 2-8 degree C
Estimated Expiration: 12 months
Shipping: Ice bag
Estimated Expiration: 12 months
Shipping: Ice bag
Related Product Information for Intracellular Fixation/Permeabilization Buffer kit
Background: Intracellular Fixation/Permeabilization Buffer Kit has been formulated and optimized for staining intracellular antigens such as cytokines and chemokines.
Product Categories/Family for Intracellular Fixation/Permeabilization Buffer kit
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Product Notes
The Intracellular Fixation/Permeabilization Buffer (Catalog #AAA178130) 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 "Intracellular Fixation/Permeabilization Buffer, 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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