Multitest cd3 fitc cd16 pe cd56 pe cd45 percp cd19 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.

For the cell subtypes of PBMC analysis, cells were collected and washed with PBS twice and then suspended in 200 μL PBS. For enumeration of mature human T (CD3⁺) cells, helper/inducer T (CD3⁺ CD4⁺) cells, cytotoxic T (CD3⁺ CD8⁺) cells, B (CD19⁺) cells, and NK (CD3⁻CD16⁺and/orCD56⁺) lymphocytes, CD3⁻FITC/CD8⁻PE/CD45⁻PerCP/CD4⁻APC reagent, BD Multitest CD3⁻FITC/CD16⁻PE⁺ CD56⁻PE/CD45⁻PerCP/CD19⁻APC reagent were used according to the manufacturer’s instructions, respectively (Cat No.340503, BD Multitest™), then quantified by flow cytometry on a FACS Calibur instrument.