Cd3 pc5

Flow cytometry was performed according to the European Pharmacopoeia (Chapter 2.7.24) using a Beckman Coulter NAVIOS (Beckman Coulter, Brea, CA, USA) [9 (link)]. Basal flow cytometry was performed at day 0 in order to evaluate the lymphocyte subpopulations on PBMCs after gradient stratification.
To initiate flow cytometry, monoclonal antibodies CYTO-STAT TetraCHROME CD45-FITC, CD56-RD1, CD19 ECD and CD3-PC5 (Beckman Coulter, Brea, CA, USA) were used for B lymphocytes and CYTO-STAT TetraCHROME CD45-FITC, CD4-RD1, CD8-ECD and CD3-PC5 (Beckman Coulter, Brea, CA, USA) for T lymphocytes. After 21 ± 3 days of expansion, flow cytometry with CD3-FITC, CD56-PE and CD45 KO (Beckman Coulter, Brea, CA, USA) was performed to evaluate CIK cell identity. As a negative control, cells were incubated without antibodies. Data were analyzed using NAVIOS software (Vs. 1.2, Beckman Coulter, Krefeld, Germany). For data analysis, we designed the physical gate as [A]. From this gate, we obtained the CD45 + CD3+ and the CD45 + CD56+ cell populations. On the dot plot we gated the double positive CIK cell population CD45 + CD3 + CD56+. On the same dot plot, we also identified the population of NK cells, CD45 + CD56 + CD3 −. The acceptance criterion of cell identity was the following: Δ% < 10% post-thaw bag vs. pre-thaw [9 (link)].

CSF and EDTA blood samples were obtained from all patients and processed as described previously (19 (link)). Briefly, cells from CSF or 100 μl EDTA blood were incubated in VersaLyse buffer (Beckman Coulter; Brea, CA) for 10 min and subsequently washed three times with PBS supplemented with 2% heat-inactivated FCS and 2 mM EDTA. Following incubation with fluorochrome-conjugated antibodies (CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange, all Beckman Coulter), cells were acquired on a Navios flow cytometer (Beckman Coulter). Analysis was conducted with Kaluza V1.2. Lymphocytes, monocytes, and granulocytes were selected based on forwards scatter channel, sideward scatter channel, CD14, and CD45 expression characteristics. Lymphocytes subsets were selected as CD3⁺CD4⁺ (T helper cells), CD3⁺CD8⁺ (cytotoxic T cells), CD3⁺HLA-DR⁺ (activated T cells), CD3⁻CD56⁺ (NK cells), CD3⁻CD19⁺ (B cells), CD3⁻CD19⁺CD138⁺ (plasma cells), whereas monocyte subsets were selected as CD14⁺CD16⁻ (classical monocytes), CD14⁺CD16⁺ (non-classical monocytes) cells.