Pacific blue conjugated anti cd3 clone sp34 2

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.

This was done according to our previously described [34 (link)]. Cells were stained with Pacific blue-conjugated anti-CD3 (Clone SP34–2, BD, Franklin Lakes, NJ), FITC-conjugated anti-CD19(clone HIB19, Biolegend, San Diego, CA), APC-conjugated anti-CD27(clone O323, Biolegend, San Diego, CA), BV605-conjugated anti-CD69(clone FN50, Bio-legend, San Diego, CA), AF700-conjugated anti-CD138(clone MI15, Biolegend, San Diego, CA), PerCP/Cy5.5-conjugated anti-CXCR5 (CloneJ252D4, Biolegend, San Diego, CA), BV510-conjugated anti-CD4 (Clone A161A1, Biolegend, San Diego, CA). After staining, cells were fixed with 2% formaldehyde-PBS (Protocol Formalin, Kalamazoo, MI) and subjected to run on a BD LSRFortessa flow cytometer (BD Biosciences, Qume Drive, San Jose, CA). Lymphocytes were gated based on forward- and side-scatters, and at least 40,000 gated events were analyzed using Summit Data Acquisition and Analysis Software (Dako Cytomation).