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.
• 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).
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.
May cause respiratory irritation.
Harmful to aquatic life.
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 INHALED: Remove person to fresh air and keep comfortable for breathing.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
If skin irritation or rash occurs: Get medical advice/attention.
IF exposed or concerned: Get medical advice/attention.
Wash thoroughly after handling.