Short Protocol Target Cells: BT-474 cells Effector Cells: PBMC Numbers of Donors 3 E:T ratio: 20:1 Incubation: 20 hours at 37°C Number of Test Concentration: 8 concentrations Dye: Calcein AM Detection Method: High-content imaging Control: hIgG1 isotype control ...
Therefore, this protocol describes methods to perform an in vitro ADCC functional assay with iRBCs. Specifically, it includes protocols on how to grow and culture iRBCs, how to culture peripheral blood mononuclear cells (PMBCs), and how to do in vitro PBMC assays to measure NK degranulation ...
NK effector cells were isolated from PBMCs using the Human NK Cell Isolation (negative selection) Kit 130–092-657 (Miltenyi Biotech, San Diego, CA), according to the manufacturer’s protocol. Median NK cell yield after isolation was typically 0.5-1 × 107 with 94–98% viability. ...
(b, c and e) PBMCs from healthy donors were used as effector cells. Percentages of Ab-specific ADCC were determined following subtraction of cell lysis mediated by control IgG from that by test antibody (5 μg/ml). (b) ADCC responses by two donors as examples and (c) Compilation...
PBMCs or enriched NK cells from healthy donors were labeled with CellTrace Violet Cell Proliferation Dye (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. After labeling, cells were cultured with 50 nmol/l concentrations of the respective drugs. Cells were harvested after 7 days...
Cryopreserved peripheral blood mononuclear cells (PBMC) obtained from an HIV-1 seronegative donor with the geographically common 158F/V Fcγ receptor IIIa phenotype were used as source of effector cells. After thawing, the cryopreserved PBMCs were rested overnight and used at an effector to ...
R (45) and ADCC assays with natural killer (NK) cells and peripheral blood monocytes (PBMC) of Fc?RIIIA genotyped donors have been described previously (12). The assay used to measure binding to human C1q has also been previously described (46). Immunofluorescence staining of purified NK ...
This model involves mixing Raji lymphoma cells with consistent numbers of NK cells, but varying number of T cells obtained from normal donor PBMCs and inoculating this mixture into NCG immunodeficient mice.2 Using this model, pre-therapy fine needle aspirates (FNAs) were collected when tumors ...
(B) CFSE labeled THP-1 cells as target cells were seeded to 96 wells Flat-bottom sterile plate, then freshly isolated human PBMCs were added as effector cell. Antibody concentration is 0.1 to 1000 nM with threefoAo SunEnlin ZhengMengshi Gao...
In secondary experiments, PBMC was incubated with lenalidomide (0/0.01/0.05/1 M, 20 min, 37°C) before the addition of ibrutinib 1 M and subsequent proceeding according to protocol as described above. Results: A significant lower rate of cell death compared to control could be observed in ...