Intracellular Cytokine Staining Protocol

Precautionary Note

1. Prior to using antibodies, do a quick spin to recover the whole volume in small vials.
2. We monitor our conjugated antibodies to make sure they are void of aggregation. Some protocols include a step of airfuging the antibody prior to multi-color flow analysis.
3. For optimal performance of fluorochrome conjugated antibodies, wrap the vials with aluminum foil and store at 4°C in the dark. Do not freeze.
4. When staining mouse antigens please note that fixation and/or delayed analysis may yield poor signal-to-noise for certain antigens. For best results, stain fresh mouse cells.

What You Need

Materials

• Directly fluorochrome-conjugated antibodies ( see charts above ) or Th1/Th2 Flow Panel
• Cells to be stained
• 12 x 75 mm round-bottom test tubes (Falcon Cat. No. 2008) or 96-well round-bottom microtiter plates

Buffers

• eBioscience IC Fixation Buffer, Cat. No. 00-8222
• eBioscience Permeabilization Buffer, Cat. No. 00-8333

Instruments

• Pipettes and pipettors
• Centrifuge
• Ice bucket or refrigerator
• Flow Cytometer

Experiment Duration

• Stimulation (variable depending on the cytokine of interest)
• 2-3 hours staining

Method

1. Prepare target cells of interest (see specific instructions).
2. Stain cell-surface antigen following the Surface Staining Protocol . The choice of the surface marker depends on the experimental question. For suggestions on best phenotypic markers for different cell types please see the appropriate BestPhenotyping Markers Chart: Mouse or Human .
3. After the last wash, fix the cells by adding 100 µl of Fixation Solution while vortexing the tube and incubate in the dark at room temperature for 20 minutes.
4. Add 1 ml of Permeabilization Buffer to each tube, centrifuge for 5 minutes and aspirate supernatant.
5. Repeat step 4.
6. Resuspend cells in 100 µl of Permeabilization Buffer and incubate in the dark at room temperature for 5 minutes.
7. Dilute the optimal concentration of fluorochrome-conjugated anti-cytokine mAb in 20 µl Permeabilization Buffer and add to the appropriate tube. Mix by vortexing. A good range of conjugated antibody for this application is 0.5-0.06 µg/10⁶ cells. eBioscience fluorochrome-conjugated anti-cytokine antibodies are also available in 20 µl/test sizes. If using these reagents, add 20 µl of pre-titrated antibody to the appropriate tube.
8. Incubate in the dark at room temperature for 20 minutes.
9. Add 1 ml of Permeabilization Buffer, centrifuge for 5 minutes and aspirate supernatant.
10. Resuspend the cell pellet in 0.5 ml of Flow Staining Buffer.
11. Run on a flow cytometer and analyze.

• RACE试剂盒
• PCR/RT-PCR/qPCR

• 电泳设备
• 紫外设备
• 普通PCR仪
• 定量PCR仪
• PCR/RT-PCR/qPCR试剂
• PCR引物
• PCR试剂
• PCR对照
• 特异性PCR试剂盒
• PCR克隆试剂盒
• RNA
• RNase检测/去除
• RT-PCR试剂
• RT-PCR标准品
• 定量PCR试剂
• 定量PCR标记
• 总RNA分离纯化盒
• PCR产物纯化