Fitc conjugated rabbit anti human igg f ab 2

EL4-huCD20, EL4, Ramos, Raji and Jurkat cells (2 × 10⁵) and PBMC from refractory low- grade B-cell lymphoma (5× 10⁵) were incubated with ICKs or mAb on ice for 30 min and washed with PBS. The binding of the antibodies was detected by incubation with FITC-conjugated rabbit anti-human IgG F(ab’)₂ (Dako, Denmark) or with PE-conjugated goat anti-mouse (Fab specific) (Abcam, USA) for 30 min on ice, followed by flow cytometry.

Cells were incubated with 14F7hT, 7C1 or isotype-matched control antibodies (10 µg/mL) on ice for 30 min and washed with PBS-BSA 1%. Binding was detected by incubation with an FITC-conjugated rabbit anti-human IgG F(ab’)₂ (Dako, Glostrup, Denmark) or a PE-conjugated goat anti-human IgG + IgM antiserum (Jackson Immunoresearch) for 30 min on ice.
For the detection of the total Neu5Gc-sialoconjugates, cells were incubated with a chicken anti-Neu5Gc antiserum (Biolegend, San Diego, CA) in binding buffer supplied by the manufacturer for 1 h on ice. After washing with cold PBS, binding was detected with an FITC-conjugated goat anti-chicken IgY antiserum (Biolegend). Chicken polyclonal IgY (Biolegend) was used as negative control.
MFI and percentage of stained cells were determined in a FACScalibur or a FACSVerse both from Becton Dickinson (Franklin Lakes, NJ) or a Gallios (Beckman Coulter, Brea, CA) cytometers. The FlowJo 10 (Tree Star, Ashland, OR) software was used for the analysis.