Victor 5 2030 multilabel plate reader

Isolated PBMCs from the whole blood samples were incubated with K562 cells to analyze the cytotoxic activity of NK cells. A whole blood sample was mixed with the same volume of RPMI medium 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA), then gently overlaid on Histopaque^®1077 (Sigma-Aldrich, Irvine, UK) and centrifuged for 20 min at 1800 rpm at 15 °C. After separation, a buffer coat layer was isolated, washed once with RPMI 1640 medium, and then resuspended in 1 mL of 10% fetal bovine serum. The isolated PBMCs (effector cell, E) were seeded into 96-well plates at ratios of 5:1 and 2.5:1 with the K562 cells (2 × 10⁴ cells/well) (target cell, T) and then incubated at 37 °C under 5% CO₂ for more than 4 h. The cytolytic activities of NK cells were analyzed via the CytoTox 96^® Non-Radioactive Cytotoxicity Assay Kit (Promega Co., Fitchburg, WI, USA) according to the manufacturer’s instructions. The color reactions were read at 490 nm using a Victor ×5 2030 multilabel plate reader (PerkinElmer, Hopkinton, MA, USA), and the results were calculated by the following formula:
$\%\text{ Cytotoxicity }=\frac{\text{Experimental-Effector Spontaneous-Target Spontaneous}}{\text{Target Maximum-Target Spontaneous}}\times 100$

WBC (lymphocytes, monocytes, and granulocytes) counts and the percentage of each component as well as platelet counts were measured using a HORIBA ABX diagnostic analyzer (HORIBA ABX SAS; Parc Euromedicine). Using these counts, the WBC to apolipoprotein A‐I ratio, monocyte to lymphocyte ratio (MLR), granulocyte to lymphocyte ratio (GLR), platelet to lymphocyte ratio (PLR), and monocyte to platelet ratio (MPR) were calculated.
High‐sensitivity C‐reactive protein (hs‐CRP), interleukin (IL)‐1β, IL‐2, IL‐6, IL‐12, tumor necrosis factor (TNF)‐α, and interferon (IFN)‐γ were measured as inflammatory markers. The hs‐CRP level was assessed with CRPHS reagent kits (Roche) and a Cobas C502 (Roche). IL‐1β, IL‐6, and TNF‐α levels were determined using Bio‐Plex Reagent kits (Bio‐Rad Laboratories) and a Luminex 200 (Luminex Corporation). IL‐2, IL‐12, and IFN‐γ levels were measured with Human IL‐2 ELISA kits (Cusabio Biotech), High Sensitivity Human IL‐12 (p70) ELISA kits (Genway Biotech Inc.), and IFN‐γ High Sensitivity Human ELISA kits (Abcam plc), respectively; the absorbance at 450 nm of the resulting reactions of the assays were evaluated using a Victor×5 2030 Multilabel Plate Reader (PerkinElmer).