转录因子比如Blimp1，T-bet在T细胞耗竭检测中经常出现，看到BD的破膜试剂说明书示意图，画的非常棒，安利一下。
1.染色前准备
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.
2.染色方法
Intracellular Staining Protocol
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.
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.
Perm/Wash: Add 2 ml of 1x Perm/Wash Buffer to the pelleted cells followed by centrifugation. Decant or aspirate wash buffer.
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.
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.
Perm/Wash: Wash cells with 2 ml 1x Perm/Wash. Centrifuge cells. Decant or aspirate wash buffer.
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.
3. 注意事项
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 5.3% formaldehyde and 1.88% methanol.
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