Multitest cd3 fitc cd8 pe cd45 percp cd4 apc reagent

The T/B/NK cells data in preoperative blood of primary OSCC patients was immediately collected and analyzed using flow cytometry. To identify and determine the percentages of mature human lymphocyte subsets in erythrocyte-lysed whole blood, including T cells (CD3⁺), B cells (CD19⁺), helper/inducer T cells (CD3⁺CD4⁺), suppressor/cytotoxic T cells (CD3⁺CD8⁺), and natural killer (NK) lymphocytes (CD3⁻CD16⁺ and/or CD56⁺), BD Multitest™ CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC reagent and BD Multitest™ CD3-FITC/CD16-PE+CD56-PE/CD45-PerCP/CD19-APC reagent were used according to the manufacturer’s instructions (Cat No. 340503, BD Biosciences, Franklin Lakes, NJ, USA), and samples were then quantified by flow cytometry on a FACS Calibur instrument. To determine the absolute counts of the lymphocyte subsets listed above, the total numbers of preoperative peripheral lymphocytes determined by the Automated Haematology Analyser XS Series (XS-1000i, Sysmex Corporation, Japan) were collected from the clinical laboratory. Since both tests came from the same batch of blood samples, we ignored the possible errors caused by the use of different detection instruments. Herein, the TBNK data of 62.3% (114/183) of OSCC patients were successfully collected and the detailed characteristic data are listed in the Supplementary Table S1.

Peripheral blood mononuclear cell (PBMC) samples were collected from patients' preoperative whole blood. For the analysis of PBMC cell subtypes, cells were collected, washed twice with phosphate‐buffered saline (PBS, Servicebio, Wuhan, PR China), and then suspended in 200‐μl PBS. BD Multitest™ CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC reagent (Cat. No. 340499, BD Multitest™, San Jose, CA, USA) and BD Multitest™ CD3 FITC/CD16 PE + CD56 PE/CD45 PerCP/CD19 APC reagent (Cat. No. 340500, BD Multitest™) were used to enumerate the CD3⁺ T cells, CD3⁺ CD4⁺ T cells, CD3⁺ CD8⁺ T cells, CD19⁺ B cells, and CD56⁺ NK cells. This was followed by quantification using a fluorescence‐activated cell sorting Calibur instrument. All study participants provided informed consent.

Peripheral blood samples collected using blood collection tubes with spray-dried K2EDTA (BD, USA) were also used for flow cytometry. All biochemical testing reagents were purchased from BD Biosciences (USA). Peripheral blood lymphocyte subsets were analyzed on the BD FACSCanto Ⅱ Flow Cytometer (BD Biosciences). BD Multitest CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC reagent (BD Biosciences) containing anti-CD3 fluorescein isothiocyanate, anti-CD8 phycoerythrin, anti-CD45 peridinin chlorophyll protein, and anti-CD4 allophycocyanin was used for identifying and determining the percentages and absolute counts of mature human T lymphocytes (CD3⁺), suppressor/cytotoxic (CD3⁺CD8⁺) T lymphocyte subsets, and helper/inducer (CD3⁺CD4⁺) T lymphocyte subsets in erythrocyte-lysed whole blood (Fig. 1). Briefly, the reagent cocktail (10 μL) was added to 50 μL EDTA-anticoagulated whole blood, and the sample was mixed and incubated for 30 min at room temperature in the dark. Erythrocytes were lysed by adding 500 μL of ammonium chloride hemolysis agent (BD Pharm Lyse, USA) for 15 min. Then the cells were washed, incubated with 2% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4), and measured on the flow cytometer^{[9 (link)]}.