Multitest 6 color tbnk reagent

Fasting whole blood from every patient was collected in EDTA collection tubes. Whole blood was incubated with BD Multitest 6-color TBNK reagent and then lysed with BD FACS™ lysing solution. Lymphocyte subpopulations were acquired and analyzed with BD FACSCanto Clinical Software. The BD Multitest 6-color TBNK reagent contains the following antibodies to identify and count different lymphocyte subsets: CD3 FITC, CD16 PE, CD56 PE, CD45 PerCP-Cy™5.5, CD4 PE-Cy™7, CD19 APC, and CD8 APC-Cy7. The results are presented as absolute count (cells/µL).

CD3⁺ T, CD4⁺ T, CD8⁺ T, CD19⁺ B, and CD16⁺ CD56⁺ NK cells were stained using BD Multitest 6-color TBNK reagent in Trucount tubes. All anti-human antibodies were purchased from BD Biosciences: CD45-APC-Cy™7 (#557833), CD3-APC (#555342), CD4-FITC (#566802), CD8-Percp-cy5.5 (#565310), CD14-AF488 (#562689), CD16-PE (#561313), CD107a-APC (#560664), CD45RA-BV605 (#562886), CD62L-PE (#555544), CD197-BV421 (#566743). Total cells were Fc-blocked and stained with indicated combinations of antibodies for 30 min on ice, then washed three times and resuspended in 1% FBS/PBS. The flow cytometric data were collected on a BD Calibur flow cytometer and analyzed using FlowJo software or Summit software.

At enrollment, three samples of venous blood (two EDTA anticoagulant tubes, one separation gel coagulation promoting tube) were collected from patients prior treatment. Fresh blood samples were delivered to our clinical laboratory within 4 hours of collection. CD3+ T, CD4+ T, CD8+ T, CD19+ B, and CD16+ CD56+ NK cells were stained using BD Multitest 6-color TBNK reagent in Trucount tubes (Cat:662997). The inhibitory and activated T lymphocyte subsets were also analyzed based on a single-platform technique by ten-color flow cytometry. The data were collected and analyzed on a BD FACS Canto II flow cytometer. The main antibodies were CD45 KrO (B36294), CD3 PB (B49204), CD4 APC-cy7 (341115), CD8 PE-cy7 (664999), PD-1 Percp-cy5.5 (561273), CD28 PE (662797), CD38 APC (345807), HLA-DR FITC (652827). The gating strategy is shown in Supplementary Figure 1. The concentrations of the cytokines IL-6 and IFN-γ were determined using enzyme-linked immunosorbent assays (Hangzhou Clongene Biotech Co. Ltd., China). The procedures were performed in accordance with the manufacturer’s protocols. Additionally, after 4-8 weeks of enrollment, the peripheral blood of patients was collected again to detect the aforementioned immune indicators and evaluate the clinical significance of any changes.

Blast formation was studied by flow-cytometric assay for specific cell-mediated immune-responses in activated whole blood (FASCIA). Heparinized blood was diluted in RPMI medium containing Glutamax I, gentamicin, and β-mercaptoethanol and stimulated with IL-2 (R&D Systems) in concentrations of 40 U/ml and 320 U/ml for 5 days. After stimulation, cells were stained with Multitest 6-Color TBNK reagent (BD) including CD3, CD45, CD4, CD8, CD19, and CD16/CD56 antibodies. After red blood cells were lysed with FACS lysing solution (BD), lymphocyte subpopulations were analyzed for the percentage of blasts based on their light scatter characteristics using flow cytometry (NovoCyte; Acea). In studying lymphocyte proliferation, purified PBMCs were incubated in culture medium (2 × 10⁵ cells per well) in 96-well polystyrene plates in the presence of increasing concentrations of IL-2 (R&D Systems) for 72 h. For the last 6 h, 1 μCi of 3H-Thymidine was added to each well. All conditions were performed in triplicates. Cells were harvested using a cell harvester (Tomtec) and thymidine incorporation was measured with beta-scintillation counter (MicroBeta; Perkin Elmer).

Each patient’s sample was placed in a BD Trucount tube that was labeled with the sample accession number. Ten microliters of BD Multitest 6-color TBNK reagent was added, followed by 50 μL of a well-mixed EDTA-anticoagulated whole-blood sample. The tube was then capped and vortexed gently to be mixed. The sample was incubated for 15 min in the dark at room temperature (20 to 25°C). Thereafter, 450 μL of 1× BD fluorescence-activated cell sorter (FACS) lysing solution was added. The sample was capped and vortexed gently and then incubated for 10 min in the dark at room temperature (20 to 25°C). The sample should have been acquired within 1 h of lysing. The sample of viable total cells was ready to be analyzed on the flow cytometer. In each sample, the levels that represent the percentages of total cell numbers of CD3⁺/CD3⁺ CD4⁺/CD3⁺ CD8⁺ T cells, CD19⁺ B lymphocytes, and CD45⁺ cells were calculated for each patient. Analysis was performed using the BD FACSCanto clinical software.