Multitest 6 color tbnk reagent kit

Heparinized peripheral blood was collected from study participants. The percentages and absolute numbers of CD4⁺ T, CD8⁺ T, CD19⁺ B and CD3^-CD56⁺ NK cells were determined by using TruCOUNT tubes and BD Multitest 6-color TBNK Reagent Kit (BD Biosciences) according to the manufacturer’s instructions. In brief, 50 μl of whole blood was labeled with 6-color TBNK antibody cocktail for 15 min in room temperature. After adding 450 μl of FACS Lysing Solution, samples were analyzed with FACSCanto flow cytometer using FACSCanto clinical software (BD Biosciences).
The following monoclonal antibodies were added to 100 μl of peripheral blood: anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD28, anti-HLA-DR, anti-CD45RA, and anti-CD45RO (BD Biosciences). Isotype controls with irrelevant specificities were included as negative controls. All of these cell suspensions were incubated for 20 min at room temperature. After lysing red blood cells, the cells were washed and resuspended in 200 μl of PBS. The percentages of CD28⁺CD4⁺ T cells, CD28⁺CD8⁺ T cells, HLA-DR⁺CD3⁺ T cells, HLA-DR⁺CD8⁺ T cells, CD45RA⁺CD4⁺ T cells, CD45RO⁺CD4⁺ T cells, CD4⁺CD25⁺CD127^- Treg cells, CD45RA⁺ Treg cells, and CD45RO⁺ Treg cells were analyzed with FACSCanto flow cytometer.

The absolute numbers of CD4⁺ and CD8⁺ T cells, B cells, and NK cells were determined by using TruCOUNT tubes and BD Multitest 6-color TBNK Reagent Kit (BD Biosciences) according to the manufacturer’s instructions.

The percentages and absolute numbers of CD4⁺ T cells, CD8⁺ T cells, B cells, and NK cells were determined using TruCOUNT tubes and BD Multitest 6-color TBNK Reagent Kit (BD Biosciences) according to the manufacturer’s instructions. In brief, 50 μL of whole blood was labeled with 6-color TBNK Ab cocktail for 15 min in room temperature. After adding 450 μL of FACS lysing solution, samples were analyzed with FACSCanto flow cytometer using FACSCanto clinical software (BD Biosciences).

Heparinized blood samples were collected from study participants at the time of notification of a positive blood culture. The absolute numbers of T, B, and NK cells were determined by using TruCOUNT tubes and the BD Multitest 6-color TBNK Reagent Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll–Hypaque density gradients. The 10 cell subsets, including CD4⁺ T cells, CD8⁺ T cells, Treg cells, T helper (Th) cells, follicular helper T (Tfh) cells, B cells, NK cells, monocytes, dendritic cells (DCs), and MDSCs (Supplementary Table S1), were detected by flow cytometry. All the staining was blocked using an Fc-blocking buffer, and isotype controls with irrelevant specificities were included as negative controls. The pellets were finally analyzed with a FACSCanto flow cytometer (BD Biosciences). The detailed antibody information is presented in Supplementary Table S2. Gating strategies for flow cytometric analysis are shown in Supplementary Figures S1–S8.

Fresh blood samples at indicated time points were collected for flow-cytometry analysis. Circulating numbers of CD4⁺ T cells, CD8⁺ T cells, CD3^-CD16⁺CD56⁺ NK cells, and CD19⁺ B cells were determined by using TruCOUNT tubes and BD Multitest 6-color TBNK Reagent Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions as previously described (Luo et al, 2021 (link)). For lymphocyte subset analysis, following antibodies were used: PerCP/Cyanine5.5 anti-human CD45 Antibody (Biolegend, 304028), FITC anti-human CD3 antibody (BD Biosciences, 561802), PE/Cyanine7 anti-Human CD4 (BD Biosciences, 560649), APC/Cyanine7 anti-Human CD8 (BD Biosciences, 557834), APC anti-human CD19 Antibody (Biolegend, 302212), PE anti-human CD16 Antibody (Biolegend, 302056), PE anti-human CD56 Antibody (Biolegend, 318306), FITC anti-Human CD38 (BD Biosciences, 567147), PerCP/Cyanine5.5 anti-Human CD27 (BD Biosciences, 560612). For CAR T-cell percentage analysis, PerCP anti-Human CD45 (BD Biosciences, 347464), APC/Cyanine7 anti-human CD3 Antibody (Biolegend, 344818) and FITC-labeled human BCMA Fc tag protein (Acrobiosystems, BCA-HF254) were used. All antibodies were used at the manufacturer’s recommended concentration. FlowJo v10 was used for analysis.