Cd27 pc7 clone 1a4cd27

Fresh PB or SF samples were processed within a few hours after sample collection. One hundred microlitres of whole PB in EDTA, or 5 × 10⁵ mononuclear cells derived from SF (separated by Ficoll-Hipaque (Cederlane, Ontario, Canada) density gradient centrifugation) in 100 μl of phosphate-buffered saline were first incubated in the dark at RT for 20 min with anti-human antibodies specific for CD45 (APC-A750) (clone J33) (dilution 1/25), CD19 (APC-700) (clone J3-119) (dilution 1/25), CD38 (PC5 or APC) (clone LS198-4-3) (dilution 1/25), CD27 (PC7) (clone 1A4CD27) (dilution 1/25) and FITC-conjugated IgD (clone IA6-2) (dilution 1/25) (all by Beckman Coulter, Marseille France). After staining, the cells were fixed, washed and erythrocytes were lysed. Samples were immediately analysed on optimally compensated 8 color Navios flow-cytometer and data were analysed with Kaluza software (Beckman Coulter, Marseille, France). Lymphocytes were gated on the basis of forward and side-scatter light properties (confirmed by CD45 staining) and at least 10,000 CD19⁺ cells were analysed. B-cell subsets were evaluated by the expression of surface B-cell markers according to IgD/CD27 classification47 (link).

Fasting venous blood (2 mL) was collected from participants in the study and control groups before surgery, and flow cytometry was performed within 24 h. Nucleated cells were stained with the following antibodies: CD3-Alexa Fluor 750 (clone UCHT1; Beckman Coulter, USA), CD4-FITC (clone 13B8.2; Beckman Coulter), CD8-Alexa Fluor 700 (clone B9.11; Beckman Coulter), CD28-PC5.5 (clone CD28.2; Beckman Coulter), CD27-PC7 (clone 1A4CD27; Beckman Coulter), CD45-Kro (clone J.33; Beckman Coulter), CD45RO-ECD (clone UCHL1; Beckman Coulter), CD95-PE (DX2; BD Biosciences, UK), and CCR7-APC (G043H7; BioLegend). The ten-color flow cytometer (Naivos; Beckman Coulter) was used for detection. The test results were analyzed using the Kaluza software (version 2.0, Beckman Coulter). The analysis scheme is illustrated in Figure 1.
Carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) were detected by an electrochemiluminescence immunoassay (ECLIA). The Roche Cobas e602 electrochemiluminescence immunoassay analyzer and its supporting reagents were used.