Description
The BD Pharmingen™ Transcription Factor Buffer Set is optimized for fixing and permeabilizing cells prior to immunofluorescent staining and flow cytometric analysis of cells that express specific intracytoplasmic and intranuclear proteins. The BD Pharmingen™ Transcription Factor Buffer Set was designed to improve ease-of-use and minimize processing time, to reduce nonspecific staining, to increase the resolution of positively stained cells and to significantly reduce cell loss during fixation, permeabilization and staining procedures. Flow cytometric detection of the many proteins known to be expressed within various intracellular compartments, especially transcription factors, is improved with BD Pharmingen™ Transcription Factor Buffer Set use. This buffer system has been found useful for fixing and permeabilizing a variety of cell types from diverse human and mouse tissues. The buffer system is flexible in supporting multiwell-plate high-throughput and bulk sample analyses and applications that require overnight sample storage. The buffer system has minimal impact on the light-scatter and autofluorescence characteristics of processed cells resulting in characteristics similar to those observed for freshly prepared, highly viable primary cells. In many cases the buffer system was found to be compatible with the immunofluorescent staining of cell-surface antigens both before and after cellular fixation and permeabilization. The buffer system is also compatible with many tandem fluorochromes.
Suggested Companion Products
Stain Buffer (FBS) RUO 500mLCat No: 554656
Stain Buffer (BSA) RUO 500mLCat No: 554657
Transcription Factor Buffer Set RUO 25TestsCat No: 562725
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Preparation and Storage
Store undiluted at 4°C.Keep stock buffers and 1x working solutions at 2-8°C. After opening the BD Pharmingen™ Transcription Factor Buffer Set, use it within six months. If stored without opening, then use before the expiration date indicated on the bottle labels.Avoid microbial contamination of reagents as incorrect results may occur.Handle TF Diluent Buffer (Component 51-9008101) in a sterile cabinet or use aseptic practices to preserve the integrity of the solution.
Product Notices
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Cy is a trademark of GE Healthcare.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Prior to intracellular staining
· Prepare single-cell suspensions from lymphoid tissues of interest (eg, human peripheral blood, mouse thymus or lymph node). Label 5-ml round-bottom 12 × 75-mm polystyrene tubes and identify appropriate antibodies for your experiment.
· Slowly invert the stock BD Pharmingen™ TF Fix/Perm Buffer (4X) and TF Diluent Buffer and TF Perm/Wash Buffer (5X) bottles 5 times before making working solutions.
· Dilute the 4x Fix/Perm Buffer using the TF Diluent Buffer to the necessary volume of 1x Fix/Perm working solution (a typical dilution for 20 tests is 5 ml of 4x Fix/Perm and 15 ml of TF Diluent Buffer). Use the 1x Fix/Perm Buffer working solution for the Intracellular Staining Protocol listed below within 1 hour of preparation.
· Dilute the 5x Perm/Wash Buffer to a 1x Perm/Wash Buffer working solution. (A typical dilution for 20 tests would be 30 ml of 5x Perm/Wash Buffer added to 120 ml of deionized water to yield 150 ml of 1x Perm/Wash Buffer). Use the 1x Perm/Wash Buffer working solution for the Intracellular Staining Protocol listed below. Store the 1x Perm/Wash Buffer at 2-8°C for up to 1 week.
· Buffers for intracellular staining should be kept on ice, or at 2-8°C, throughout the Intracellular Staining Protocol.
· Surface Staining: Prepare cell suspension containing 10^e6 cells per ml in flow cytometry stain buffer, such as BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) or Stain Buffer (BSA) (Cat. No. 554657). Incubate 100 µl of cells per tube with fluorescent antibodies (eg, antibodies specific for CD4, CD8, CD25, CD19) for 30 minutes at 2-8°C. Wash one time with 2 ml of stain buffer and centrifuge cells at 350g for 5 minutes before beginning the intracellular staining protocol listed below.
Intracellular Staining Protocol
1. Fix/Perm: After the cell surface staining procedure is completed, aspirate residual stain buffer and loosen the cell pellet by vortexing briefly. Add 1 mL of freshly prepared 1x Fix/Perm Buffer working solution to each tube and resuspend cell pellets by vortexing for approximately 3 seconds. Incubate samples at 2-8°C for 40-50 minutes protected from light.
2. Perm/Wash: Add 1 ml of 1x Perm/Wash Buffer directly to the fixed and permeabilized cells suspended in the 1x Fix/Perm Buffer. Pellet the cells by centrifugation. (Note: All centrifugation steps post Fix/Perm are at 350g and at 2-8 °C for 6 minutes). Decant or aspirate the supernatants.
3. Perm/Wash: Add 2 ml of 1x Perm/Wash Buffer to the pelleted cells followed by centrifugation. Decant or aspirate wash buffer.
4. Intracellular Staining: Add 80-100 µl of 1x Perm/Wash Buffer to cell samples and the fluorescent antibodies specific for intracellular proteins (eg, FoxP3, T-bet and/or IL-17A) and for nonspecific control staining (eg, matching fluorescent Ig isotype controls) to each tube. Vortex tube or rack for 10 seconds and incubate at 2-8°C for 40-50 minutes protected from light.
5. Perm/Wash: Briefly vortex samples prior to washing. Wash cells with 2 ml of 1x Perm/Wash Buffer. Centrifuge cells. Decant or aspirate the wash buffer.
6. Perm/Wash: Wash cells with 2 ml 1x Perm/Wash. Centrifuge cells. Decant or aspirate wash buffer.
7. Sample preparation for flow cytometry: Resuspend cell pellet in 350 µl of flow cytometry stain buffer. Analyze the cells and acquire data using a flow cytometer.
Notes:
· Due to the fixation and permeabilization procedure, forward and side light-scatter signals will be slightly different than those of live cells.
· The buffer system is optimized for use with fluorescence settings established by using the BD™ Cytometer Setup & Tracking Beads Kit (Cat. No.
642412). However, for your application, minor adjustments in gate and/or detector voltage may need to be made prior to compensation and acquisition.
· Target the acquisition for a statistically significant number of events.
· A titration of the fluorescent antibody"s optimal staining amount and optimization of the staining time may be required in your application.
Danger:TF Fix/Perm Buffer (4X) (Component 51-9008100) contains 5.37% formaldehyde (w/w), 2.20% 2-(2-Ethoxyethoxy)ethanol, and 1.89% methanol (w/w).
Hazard statements:
Harmful if swallowed or in contact with skin.
Causes skin irritation.May cause an allergic skin reaction.
Causes serious eye damage.
Suspected of causing genetic defects.
May cause cancer.
Harmful to aquatic life.
Precautionary statements:
Obtain special instructions before use. Do not handle until all safety precautions have been read and understood.
Wear protective clothing / eye protection/ gloves.Contaminated work clothing should not be allowed out of the workplace.
Avoid breathing mist/vapours/spray.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.
Continue rinsing.
IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. Immediately call a POISON CENTER/doctor.
IF ON SKIN (or hair): Take off immediately all contaminated clothing. Wash with plenty of water.
If skin irritation or rash occurs: Get medical advice/attention.
IF exposed or concerned: Get medical advice/attention.
Wash thoroughly after handling.
Caution: TF Perm/Wash Buffer (5X) (Component 51-9008102) contains ≤0.09% sodium azide.Sodium azide yields highly toxic hydrazoic azid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
Safety Data Sheets (SDS) can be found at our website: regdocs.bd.com
