Rabbit complement ma

The complement-dependent cytotoxicity assay (CDC) was performed with IgG purified from pre-immunized (day 1) and post-immunized (day 126) sera of cynomolgus monkeys using Zeba™ Spin Desalting Columns (Thermo Scientific) and the Melon Gel IgG Purification Kit (Thermo Scientific). CDC activity was evaluated in Jurkat-GFP cells and MCF-7, SHIN3 and OVCAR cells labeled with 5 μM of CFSE (carboxyfluorescein succinimidyl ester, Life Technologies). Cells (6000/well) were incubated with cynomolgus antibodies at various concentrations and DRAQ7 viability dye (Abcam) at 2 μM, in the presence or absence of 1 % of rabbit complement-MA (Cedarlane) for 2 h in 384-well plates (37 °C). Green fluorescence (live cells) and red (dead cells) fluorescence were measured on an Incucyte™ ZOOM instrument (Essen Bioscience), and the number of cells was calculated using ZOOM software (Essen Bioscience). All measurements were performed in duplicate. The percentage of CDC was determined as follows: % of CDC = ((% of dead cells)^complement – (% of dead cells)^medium) normalized against the control without antibody.

ICON-CDC to CD133+ CSCs and CD133- non-CSCs was determined by a fluorescent dye Calcein AM release assay, as described [21 (link)]. Briefly, the CD133+ CSCs and non-CSC CD133- cells were seeded in 96-well U-bottom microplates overnight. The next morning the cells were labeled with 4 μM Calcein AM (Molecular Probes) for 40 min and then the cells were incubated with ICON diluted in 1× VBS buffer supplemented with 10mM CaCl₂ and followed by incubation with 1:4 diluted rabbit complement MA (Cedarlane Laboratories) as a source of complements. Controls include ICON alone and complement alone, buffer alone (as spontaneous release control) and maximal release control (0.1% Triton X-100 lysis). After 4-hr incubation, the plate was briefly centrifuged and supernatant was transferred to a new 96-well black plate with clear, flat bottom. Fluorescence in supernatants was read on a fluorescence microplate reader (Molecular Devices). CDC cytotoxicity was calculated as described [21 (link)].