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 five times the stock BD Pharmingen™ TF Fix/Perm Buffer (4X), TF Diluent Buffer and TF Perm/Wash Buffer (5X) bottles 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 an 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 one 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 million 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 cells one time with 2 ml of stain buffer and move forward with the intracellular staining protocol listed below.
Intracellular Staining Protocol
1. Fix/Perm: After the cell surface staining procedure is completed, aspirate residual stain buffer. Resuspend cell pellets by brief vortexing and add 1 ml of freshly prepared 1x Fix/Perm Buffer working solution to each tube. Vortex samples for approximately three seconds after adding the Fix/Perm Buffer. 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
• 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 51-9008314) contains 5.0% formaldehyde (w/w) and 1.76% methanol (w/w).
Harmful if swallowed or in contact with skin.
Toxic if inhaled.
Causes skin irritation.
Causes serious eye damage.
May cause an allergic skin reaction.
Suspected of causing genetic defects.
May cause cancer. Route of exposure: Inhalative.
May cause respiratory irritation.
Wear protective clothing / eye protection.
Wear protective gloves.
Avoid breathing mist/vapours/spray.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.
IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
IF ON SKIN: Wash with plenty of water.
Caution: TF Perm/Wash Buffer (5X) (Component 51-9008316) 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.