Mouse Regulatory T Cell Multi-Color Flow Kit

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, mouse regulatory T cells are assessed by flow cytometry using the following procedure:

• Wash and resuspend cells in Flow Cytometry Staining Buffer
• Stain cells with cell surface marker fluorochrome-conjugated antibodies or isotype controls
• Wash and resuspend cells in Flow Cytometry FoxP3 Staining Buffer
• Stain cells with FoxP3-APC antibody or isotype control
• Wash cells and resuspend in Flow Cytometry Staining Buffer
• Analyze samples by flow cytometry

View Graphic Procedure

 

Reagents Supplied in the Mouse Regulatory T Cell Multi-Color Flow Cytometry Kit (Catalog # FMC014)

• CD25-PE (Clone 280406; rat IgG_2A)
• CD4-PerCP (Clone GK1.5; rat IgG_2A)
• FoxP3-APC (Goat IgG)
• Goat IgG-APC Isotype Control
• Flow Cytometry FoxP3 Staining Buffer (with 1% formaldehyde and 0.09% sodium azide)
• Flow Cytometry Staining Buffer (with BSA and 0.09% sodium azide)
• Includes enough reagents to perform 50 assays

• PBS or Hanks’ Balanced Salt Solution (HBSS)

Cell Staining Protocol with Simultaneous Fixation/Permeabilization

1. Harvest cells and wash 2 times with PBS or HBSS.
2. Resuspend cells in Fixation Cytometry Staining Buffer (approximately 1 x 10⁶ cells/100 µL) and transfer to 5 mL flow cytometry tubes.

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3. Add 10 µL CD25 and CD4 fluorochrome-conjugated antibodies.
4. Incubate the samples at room temperature for 30-45 minutes in the dark.

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5. Wash the cells in Flow Cytometry FoxP3 Staining Buffer.

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6. Add 10 µL FoxP3-APC antibody or goat IgG Isotype Control.
7. Incubate the samples at room temperature for 1 hour in the dark.

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8. Wash the cells in Flow Cytometry FoxP3 Staining Buffer.
9. Resuspend the cells in 200-400 μL of Flow Cytometry Staining Buffer.

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10. Analyze the expression of regulatory T cell markers simultaneously by flow cytometry.