Lsr 2 device

Splenocytes obtained from humanized mice were dissociated and incubated with a hypotonic solution (0.83% ammonium chloride/20 mM HEPES, pH 7.2, for 5 min at RT) to lyse the erythrocytes. Immune staining of the B cells was performed essentially as described [15 (link)] after a blocking step and staining with fluorochrome-labeled antibodies. Anti-human CD45-Pacific blue (Biolegend, 304022); p27-287 (gB) (kindly provided by Prof. Michael Mach, Erlangen); Anti-mouse IgG-Alexa647 (Biolegend, 405322); Anti-mouse IgG-Alexa488 (Biolegend, 405319); p63-27 (iE1) (kindly provided by Prof. Michael Mach, Erlangen); anti-mouse-IgG-Alexa647 (Biolegend; 405322); anti-human IgG -Alexa647 (Jackson ImmunoResearch, 109-605-003). Flow cytometry acquisition was performed with a LSR II device (BD Biosciences, Heidelberg, Germany). Data analysis was performed with FlowJo (Treestar Inc., Ashland, OR, USA).
For the detection of gp350-specific human IgG antibodies, parental PCI-1 cells and PCI-1 cells stably expressing gp350 were incubated with supernatants from LCLs, followed by incubation with a anti human-IgG Alexa647-labeled secondary antibody.

To exclude dead cells, the Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) was used. NY-ESO-1-specific TCR expression was evaluated using the PE-conjugated A0201 NY-ESO-1-tetramer (kindly provided by Hiroshi Shiku, Mie University, Tsu, Japan). The following fluorochrome-conjugated antibodies were used for immunophenotyping of different surface markers on SW982 cells as well as NY-ESO-1-specific T cells: anti-CD80-APC (BioLegend, San Diego, CA, USA), anti-HLA-ABC-FITC (eBioscience, San Diego, CA, USA), anti-programmed cell death protein ligand 1 (PD-L1)-PE (eBioscience), anti-CD3-V500 (AmCyan) (BD Biosciences, San Diego, CA, USA), anti-CD8-Parcific blue (BioLegend), anti-CD25-PE-Cy7 (BioLegend), anti-CD4-Alexa Fluor 700 (eBioscience). Data acquisition was performed on an LSR II device (BD Biosciences) and data were analyzed using the FlowJo software (TreeStar, Ashland, OR, USA).

The frequency of CMV-specific CD8⁺ T cells was determined as described earlier [30 (link)]. The acquisition was performed on an LSRII™ device (BD Biosciences, San Diego, CA, USA), and the analysis was performed using the BD FACSDiva™ software (BD Bioscience, San Diego, CA, USA).

Immunophenotyping and immunomonitoring were performed on rested PBMCs except MDSCs (18 (link)). Cells were stained with different combinations of antibodies (Supplementary Table 1). Blocking buffer containing 50% human serum was used to reduce nonspecific binding, and NEAR-IR was used for dead cell exclusion. Each antibody was first titrated to determine its optimal concentration for staining. Appropriate negative controls, fluorescence minus one (FMO) control or un-stimulated control, were used in the study. All acquisitions were performed on an LSRII device (BD Biosciences). To ensure the quality of measurement, CS&T was performed per working day. Furthermore, fluorescence compensation was applied before data acquisition. Data were analyzed using BD FACSDiva software (BD Biosciences). Gate placement was based on the recommendation from the International Multiconsortia Proficiency panel (19 (link), 20 (link)).

The quality and quantity of expression of different markers were determined by multiparametric flow cytometry. Samples were stained by different combinations of antibodies against CD3, CD4, CD8, CD11b, CD14, CD16, CD19, CD27, CD56, CD57, CD62L, CD107a, CD159c (NKG2C), CD314 (NKG2D), IFN-γ, and TNF-α. Detailed information of antibody is shown in Supplementary Table 1. 7-Amino actinomycin D (7AAD) or Near-infrared (NEAR-IR) was used for live/dead cell discrimination. Fluorescence minus one, unstimulated, and autofluorescence controls were included in order to place the gate more accurately. To reduce the variation, samples from the same patient at different time points have been analyzed on the same day. The Fc receptors were blocked by blocking buffer A [50% fluorescence-activated cell sorting buffer (FACS) + 50% human serum] or blocking buffer B (50% perm buffer + 50% human serum) prior to surface marker staining or intracellular cytokine staining, respectively. All acquisitions were performed on a LSRII device (BD Biosciences) and the data were analyzed by FACS Diva software (BD Biosciences). The cellular division index was determined by Flowjo software (TreeStar).