Rabbit anti human ig fitc

Cell surface binding: HEK cells expressing human or rat P2X3, or human P2X2, and control cells were incubated with antibody supernatants for 1 h on ice, washed and incubated with rabbit anti-human Ig FITC (Sigma) for 1 h on ice, washed and analyzed by flow cytometry.
Inhibition of Ca⁺⁺ signaling: Antibodies were tested for ability to inhibit α,β-meATP-induced calcium flux using a cell-based Ca²⁺ flux assay [41 (link)]. Briefly, canine Cf2Th cells were transfected with the appropriate expression vector in poly-lysine coated, black 384-well plates with clear bottoms (Costar). Cells were incubated for 22 h at 37°C then washed twice and loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit, Molecular Devices), incubated for 1.5 h, then moved to a Flexstation II-384 (Molecular Devices) set at 32°C. After a 10-min temperature equilibration, the specific P2X3 agonist α,β-meATP (Sigma) was injected (at t = 20 s) and fluorescence was measured for 60 s (reading every 3 s). Data sets were analyzed using Prism 5.0 software (GraphPad Software, Inc).

Cell lines: Cf2Th/synCCR5 cells, derived from the canine thymocyte cell line Cf2Th, were obtained from the NIH AIDS Research and Reference Reagent Program and were cultured in DMEM, 10% FBS, 1× Glutamate, 1× NEAA, 1× Pen/strep, 500 µg/ml G418, 500 µg/ml zeocin and 3µg/ml puromycin (Invitrogen). CCR5-transduced SupT1-M10 [39 (link)] and parental SupT1 (not transduced with CCR5) were kindly provided by H. Garg.
Cell surface binding: CCR5 expressing and non-expressing control cells were incubated with antibody supernatants for 1 h on ice, washed and incubated with rabbit anti-human Ig FITC (Sigma) for 1 h on ice, washed and analyzed by flow cytometry.
Long-lasting internalization of CCR5: Receptor internalization was induced by exposing SupT1-M10 cells to supernatants containing mAbs to CCR5 for 48 h at 37°C. To verify receptor downregulation, cells were also incubated with Mip1beta (CCR5 ligand) for 1 h. As positive control of long-lasting internalization, a previously described pool of mouse antisera to CCR5 was used [40 (link)].