Cyto stat tetrachrome cd45 fitc cd4 rd1 cd8 ecd cd3 pc5

EDTA blood was analysed by the automated Sysmex system to determine the frequencies of basic blood cell types including lymphocytes, eosinophils and neutrophils. For the flow cytometry, EDTA blood was stained with the following antibodies: CYTO‐STAT tetraCHROME CD45‐FITC/CD4‐RD1/CD8‐ECD/CD3‐PC5, CYTO‐STAT tetraCHROME CD45‐FITC/CD56‐RD1/CD19‐ECD/CD3‐PC5 (Beckman Coulter), anti‐CD16‐PE (3G8, BD Bioscience), CD25‐RD1 (ACT‐1, Dako‐Agilent), HLC‐DR‐ECD (IO/Immu‐357, Beckman Coulter) and CD23‐RD1 (MHM6, Dako‐Agilent). After 15‐min incubation at room temperature in the dark, the samples were lysed which was followed by the paraformaldehyde fixation using the Coulter Q‐Prep Workstation with IMMUNOPREP reagent system (Beckman Coulter). Isotype controls had the equal protein concentration as the test antibodies. Four‐colours flow cytometry was preformed using an EPICS™ XL‐MCL (Beckman Coulter) using the software Expo™ 32 version for data acquisition and evaluation.

Flow cytometry was performed according to European Pharmacopoeia (Chapter 2.7.24) using Beckman Coulter NAVIOS (Beckman Coulter, Brea, CA, US) [19 ]. At Day 0, before PBMCs seeding, basal flow cytometry was performed in order to evaluate the lymphocyte subpopulations. About 0.5–1 × 10⁶ cells were incubated for 20 min, at 4 °C in the dark with respectively CYTO-STAT TetraCHROME CD45-FITC, CD56-RD1, CD19 ECD, CD3-PC5 (Beckman Coulter, Brea, CA, US) for B lymphocytes and CYTO-STAT TetraCHROME CD45-FITC, CD4-RD1, CD8-ECD, CD3-PC5 (Beckman Coulter, Brea, CA, US) for T lymphocytes. Cells were washed and re-suspended in 300 µl of PBS. After 20–21 days of expansion flow cytometry was performed to evaluate the CIKs identity. Briefly, 0.5–1 × 10⁶ cells were incubated as previously described with CD3-FITC, CD56-PE, CD45 KO (Beckman Coulter, Brea, CA, US). As negative control, 0.5–1 × 10⁶ cells were incubated without antibody. For data analysis, we designed the physical gate as [A]. From this gate, we obtained the CD3+ and the CD56+ cell population. In the dot plot CD3xCD56 we gated the CIK double positive CD3+CD56+ cell population. The tail is representative of the primitive CD3+CD56+. These cells were the cellular therapy product as it was in compliance with the identity acceptance criteria.