This was done as we previously described [24 (link), 33 (link), 44 (link)]. Cells were harvested at day 7 after culture and stained with Pacific blue-conjugated anti-CD3 (Clone SP34-2, BD, Franklin Lakes, NJ), PE conjugated anti-CD4 (clone OKT4, eBioscience, San Diego, CA), FITC-conjugated Vγ2 (Clone 7A5, Pierce, Rockford, IL) and purified Vδ2 (clone 15D, Pierce) in combination of allophycocyanin-conjugated goat anti mouse IgG (Dako, Carpinteria, CA). After staining, cells were fixed with 2% formaldehyde-PBS (Protocol Formalin, Kalamazoo, MI) and subjected to run on a CyAn ADP flow cytometer (DakoCytomation, Carpinteria, CA). Lymphocytes were gated based on forward- and side-scatters, and pulse width and at least 40 000 gated events were analyzed by using Summit Data Acquisition and Analysis Software (Dako Cytomation). Further special gates and quadrants for data analysis were determined on nonstaining, specific antibody staining, and isotype control antibody background staining.
Protocol detail
Flow Cytometry Analysis of T-cell Subsets
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Pacific blue-conjugated anti-CD3 (Clone SP34-2,
CyAn ADP flow cytometer
Summit Data Acquisition and Analysis Software
Allophycocyanin
Anti cd3
Anti igg
Antibody
Cells
Clone
Fitc
Formaldehyde
Formalin
Goat
Isotype
Lymphocytes
Mouse
Pulse
Corresponding Organization : Illinois College
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Variable analysis
independent variables
- Cells were harvested at day 7 after culture
- Percentage of CD3+ cells
- Percentage of CD4+ cells
- Percentage of Vγ2+ cells
- Percentage of Vδ2+ cells
- Cells were stained with Pacific blue-conjugated anti-CD3 (Clone SP34-2, BD, Franklin Lakes, NJ)
- Cells were stained with PE conjugated anti-CD4 (clone OKT4, eBioscience, San Diego, CA)
- Cells were stained with FITC-conjugated Vγ2 (Clone 7A5, Pierce, Rockford, IL)
- Cells were stained with purified Vδ2 (clone 15D, Pierce)
- Cells were stained with allophycocyanin-conjugated goat anti mouse IgG (Dako, Carpinteria, CA)
- Cells were fixed with 2% formaldehyde-PBS (Protocol Formalin, Kalamazoo, MI)
- Cells were analyzed using a CyAn ADP flow cytometer (DakoCytomation, Carpinteria, CA)
- Lymphocytes were gated based on forward- and side-scatters, and pulse width
- At least 40,000 gated events were analyzed using Summit Data Acquisition and Analysis Software (Dako Cytomation)
- Nonstaining controls
- Specific antibody staining controls
- Isotype control antibody background staining
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