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)].
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)].
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