About 20 × 106 PBMC were depleted of CD14+ monocytes by Dynabeads® CD14 (Invitrogen) according to manufacturer’s instructions.

CFSE-labelled CD14+ monocytes were added to the CD14-depleted PBMC to reconstitute a PBMC population with CFSE-labelled CD14+ monocytes. The reconstituted PBMC were stained with anti-CD14 PE, HLA-DR-PE, CD1a-PECy5.5, with anti-CD40-PECy5.5, CD80-PECy5.5, CD- 83-PECy5, CD86-PECy5.5 and with anti-HLA-A,B,C-PECy5.5 (eBioscience) for tracing the phenotype of CFSE-labelled CD14+ cells during CD3 stimulation or during the CAPRI procedure. Flow cytometry.  Expression of cell surface markers was determined by flow cytometry using the Becton-Dickinson FACScan analyzer and CellQuest software (Becton-Dickinson). CD14+ cells were CFSE-labelled to trace the changes

in phenotype. In brief, cells were harvested and stained with anti-CD14 PE, HLA-DR-PE, CD1a-PECy5.5, Selisistat research buy with anti-CD40-PECy5.5, CD80-PECy5.5, CD 83-PECy5, CD86-PECy5.5 and with anti-HLA-A,B,C-PECy5.5 to trace the phenotype of CFSE-labelled CD14 cells during CD3 or CAPRI stimulation. For this website the analyses of cell surface markers on CD3-stimulated and CAPRI cells, cells were collected and stained with anti-CD3-FITC, CD14-PE, CD19-PECy5.5, with anti-CD3-FITC, CD4-PE, CD8-PECy5.5, with anti-CD3-FITC, CD14-PE, CD56-PECy5.5 and with anti-CD3-FITC, CD16-PE, CD56-PECy5.5. For Foxp3 staining, cells were stained first with anti-CD4-PE, fixed, permeabilized with human Foxp3 staining buffer set and then stained with FITC-anti-human Foxp3. The conjugated mouse monoclonal antibodies were obtained from BD Biosciences or eBioscience. The human Foxp3 staining buffer set was obtained from eBioscience. Presence of CD4+ T lymphocytes could not be replaced in the priming phase or in the cytotoxicity assay by supernatants from CAPRI cell cultures.  CAPRI culture supernatants were added to CAPRI cell cultures to clarify whether CD4+ T lymphocytes provided only ‘cytokine help’ to cytotoxic CD8+ T cells

or participated as effector cells in cancer cell destruction. To avoid the depletion of CD14+CD4+ Diflunisal monocytes, CD3+ cells were first isolated from PBMC cultures (1), and then CD4+ cells were depleted. The CD4+-depleted CD3 isolate was added to (1). Supernatants were added before CD3 activation or to unstimulated PBMC, which were added in the second step to supply T cells expressing the αβTCR. Cytotoxicity testing of human CAPRI cells against autologous breast cancer cells in nude mice.  Animal experiments were authorized by the ethic committee of the University of Wuhan, China, and designed by S. Gu and performed at the Wuhan University under the supervision of S. Gu. Twelve 6-week-old nude female mice (BALB/c-nu) were obtained from Wuhan University, Center for Animal Experiments, China.