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Product Information

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Contents: Ready-SET-Go! Intracellular Cytokine Staining Kit Catalog Number: 88-7777 Storage Conditions: Store at 4 degrees C. DO NOT FREEZE. LIGHT SENSITIVE MATERIAL.

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Description

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The eBioscience Ready-SET-Go! IC Staining Kit is a flexible, customizable collection of reagents and buffers for intracellular staining and flow cytometric analysis of 1, 2, or 3 different cytokines. The kit includes monensin (protein transport inhibitor for inclusion in tissue culture medium during cell activation), fixation buffer, permeabilization buffer, and your choice of one, two, or three each of the following: fluorochrome conjugated anti-cytokine antibody, corresponding fluorochrome-labelled isotype control, and unlabelled anti-cytokine antibody (blocking control). The fluorochrome-conjugated antibody reagents are pre-diluted and sufficient for 25 tests each.

Components:

1. Fluorochrome-conjugated anti-cytokine antibody: 1 vial per cytokine, enough for 25 tests
2. Purified mAb (blocking mAb): 1 vial per cytokine, enough for 25 tests
3. Fluorochrome-conjugated isotype control antibody: 1 vial per cytokine, enough for 25 tests
4. Permeabilization buffer: 100 ml per cytokine, working solution
5. Fixation buffer: 25 ml per cytokine, working solution
6. Monensin: 0.2ml per cytokine, 1000X concentrated

What You Need

Materials

• Cells to be stained
• 12 x 75 mm round-bottom test tubes (Falcon Cat. No. 2008) or 96-well round-bottom microtiter plates

Instruments

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

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. Add 20 ul of pre-titrated fluorochrome-conjugated anti-cytokine mAb to the appropriate tube. Mix by vortexing.
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 eBioscience Flow Cytometry Staining Buffer (Cat. 00-4222)
11. Run on a flow cytometer and analyze.