Quantitative data was obtained from spectrophotometry (antioxidant SOD and catalase activity) and flow cytometry (cell cycle progression and mitochondrial membrane potential). Three independent repeats were performed for all experiments where the averages and the standard deviations were determined. Averages are demonstrated using bar charts and standard deviations are displayed with errors bars. A p-value < 0.05 calculated by means of the Student t-test was utilized for statistical significance and is specified using an asterisk (*). Flow cytometry analysis includes at least 10,000 events. The percentage of cells were calculated that represented polarized and depolarized cells within the histogram relative to the histogram of cells propagated in complete growth medium using the statistics provided by Kaluza analysis software version 2.0 software from Beckman Coulter Life Sciences (Indianapolis, IN, USA). Cell cycle distributions were quantified with Kaluza analysis software version 2.0 software from Beckman Coulter Life Sciences (Indianapolis, IN, USA) by assigning relative DNA content per cell to sub-G1, G1, S and G2/M fractions using Kaluza analysis software version 2.0 software from Beckman Coulter Life Sciences (Indianapolis, IN, USA).
Kaluza analysis software version 2
Manufactured by Beckman Coulter
Sourced in United States
Kaluza Analysis software, version 2.1, is a data analysis application designed for use with Beckman Coulter flow cytometry instruments. The software enables users to visualize, analyze, and interpret data generated from flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Gallios cytometer, by Beckman Coulter (3 mentions)
Gallios flow cytometer, by Beckman Coulter (3 mentions)
Apc antihuman cd45 clone hi30, by BD (2 mentions)
Bv421 antihuman cd66b clone g10f5, by BD (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Image lab version 6, by Bio-Rad (2 mentions)
Cytofix cytoperm reagent, by BD (2 mentions)
Fitc conjugated anti np antibody, by Merck Group (2 mentions)
Perm wash, by BD (2 mentions)
Cellquest software, by BD (1 mentions)
Anti cd3 ecd, by Beckman Coulter (1 mentions)
Anti cd4 bv510, by BD (1 mentions)
Cytofix, by BD (1 mentions)
Gallios analyzer, by Beckman Coulter (1 mentions)
Cd38 apca750, by Beckman Coulter (1 mentions)
Navios 10 color flow cytometer, by Beckman Coulter (1 mentions)
Recombinant mouse ifn γ, by BioLegend (1 mentions)
Facsaria 3 fusion, by BD (1 mentions)
Compbead, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
27 protocols using kaluza analysis software version 2
1
Antioxidant Activity and Cell Cycle Impacts
2
ILT-2 and B Cell Phenotyping in Spleen
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To study ILT-2 surface expression on splenic B cells, we stained the cell suspension with anti-human CD19 (APC, clone SJ25C1, eBioscience, Frankfurt Germany) and anti-human ILT-2 (PE, clone GHI/7, BD Bioscience, Heidelberg, Germany). For surface staining of CD19, CD27, and CD38 on splenic B cells, the following monoclonal antibodies were applied: anti-human CD19 (PE, clone HIB19), anti-human CD27 (FITC, clone O323), and anti-human CD38 (APC, clone HIT2). All antibodies were provided by eBioscience. Isotype-matched antibodies served as negative controls (BD Bioscience). Stained samples were assessed in a FACSCalibur analysis, data acquisition was performed with CellQuest software (BD), and data were analyzed by Kaluza Analysis Software Version 2.1 (Beckman Coulter, Brea, California USA). The expression of antigens is given as the index of mean fluorescence intensity (MFI) was calculated as follows: (MFI sample–MFI IgG control)/MFI IgG control.
3
Quantification of NF-κB Activation in Immune Cells
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PBMC suspensions were transferred to a V-bottom plate while pooling the duplicates. Following centrifugation for 2.5 min, cell surface markers were stained in the dark for 30 min at 4 °C with a monoclonal antibody mix containing anti-CD3-ECD (1:25; Beckman Coulter, Brea, CA, United States), anti-CD4-BV510 (1:50; BD Bioscience, Franklin Lakes, NJ, United States), anti-CD8-APC Alexa Fluor 700 (1:400; Beckman Coulter), and anti-CD14-FITC (1:50; Dako; Agilent Technologies). Subsequently, cells were washed twice with flow cytometry buffer (FCM buffer, 0.2% BSA in PBS) and fixed (BD Biosciences Cytofix, 554655) for 10 min at 37 °C. Next, cells were washed and permeabilised with perm buffer IV (1:10 diluted with PBS, BD Biosciences Phosflow, 560746) for 20 min on ice in the dark. Cells were then stained intracellularly with anti-NF-κB p65 (pS529)-PE antibody (1:50; eBioscience; Thermo Fisher Scientific, Inc, Waltham, MA, United States) for 20 min at 4 °C. After washing the cells twice in FCM-buffer, the suspensions were measured on a Beckman Coulter Navios EX Flow Cytometer using Navios System Software. Cell immunophenotypes were analysed using Kaluza Analysis Software version 2.1 (Beckman Coulter). The mean fluorescent intensities (MFIs) were calculated using the median pNF-κB p65 expression levels within the gated immune cell populations of interest.
4
Measuring Mitochondrial Membrane Potential
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Growing fibroblasts were washed with PBS and incubated with 50 nm tetramethylrhodamine, ethyl ester (TMRE) (Molecular Probes) in prewarmed PBS for 30 minutes at 37°C. Cells were trypsinized, centrifuged at 1,000g for 5 minutes, and resuspended in prewarmed PBS. All samples were captured by a Gallios Analyzer (Beckman Coulter), which recorded 20,000 cells for each genotype tested. FCCP (200 μM for 10 minutes) was used as a positive control. Histograms showing the inner Ψm levels were obtained after gating live cells. The data were analyzed with Kaluza Analysis software, version 2.1. (Beckman Coulter).
5
Multiparametric Flow Cytometry of B Cells
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Peripheral blood mononuclear cells (PBMC) were isolated from whole blood samples using density gradient centrifugation. PBMC viability was assessed by trypan blue exclusion and cells were resuspended in phosphate buffered saline (PBS) at 20×106/mL. PBMC (100 mcL of the suspension) were added to a Duraclone IM B cell 8-color antibody panel tube containing the following stabilized antibodies: (CD45-KrO (Krome Orange), CD19-ECD (Electron Coupled Dye), IgD-FITC (Fluorescein isothiocyanate), CD27-PC7 (Phycoerythrin-cyanine 7), CD21-PE (Phycoerythrin), CD24-APC (Allophycocyanin), CD38-APC-A750 (Allophycocyanin-750), and IgM-PB (Pacific Blue) (Beckman Coulter, San Jose, CA). Following a 20-minute incubation at room temperature, protected from light, the cells were washed with PBS, fixed and data acquired on a Navios 10-color flow cytometer (Beckman Coulter). Data were analyzed using Kaluza Analysis Software, version 2.1 (Beckman Coulter).
6
Phagocytosis Assay of Malaria Merozoites
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Leukocytes were transferred to 96-well U-bottom plates containing 6 × 104 cells in 100 µl of cell medium per well. Opsonized, ethidium bromide-stained merozoites (4–6 × 105 per well) were washed twice with FACS buffer, resuspended in cell medium, and 100 µl were added to each leukocyte containing well. After an incubation of 30 min (unless stated otherwise) at 37 °C and 5 % CO2, plates were centrifuged in a prechilled centrifuge and washed twice with ice-cold FACS buffer to stop phagocytosis. Cells were resuspended in 200 µl of cold FACS buffer and incubated for 1 hour at 4 °C with 1:1600 FITC antihuman CD14 (clone TuK4; Thermo Fisher Scientific MA1-82074), 1:400 BV786 antihuman CD16 (clone 3G8; BD Biosciences 563690), 1:800 APC antihuman CD45 (clone HI30; BD Biosciences 555485), and 1:800 BV421 antihuman CD66b (clone G10F5; BD Biosciences 562940) antibodies. After washing thrice with FACS buffer, sample fluorescence was quantified with a CytoFLEX S (Beckman Coulter Life Sciences). Phagocytosis was determined by measuring the ethidium bromide fluorescence using the 610/20 nm detector. Data analysis were performed with Kaluza Analysis Software version 2.1 (Beckman Coulter Life Sciences). The gating strategy used is shown in Supplementary Figure 2 .
7
Quantification of Intracellular ROS Levels
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Cytosolic ROS levels were determined using the oxidation-sensitive fluorescent probe DCFH-DA. Intracellular ROS oxidize this probe to a highly fluorescent compound, DCF. Following treatments with the specified compounds at the indicated time points, cells were washed with HBSS containing 10 mM HEPES buffer (pH = 7.4). Subsequently, cells were stained with 5 μM DCFH-DA for 15 min at 37 °C in the dark using a shaking water bath and washed with HEPES-buffered HBSS. For the positive control, DCFH-DA-loaded cells were treated with 0.5 mM H2O2 for 15 min. Untreated and unstained cells served as the negative control. In the experiment reported in Figure 5 c,d, DCFH-DA-loaded cells were divided into two groups and incubated with vehicle (HBSS) or 10 µM of H2O2 for 15 min, followed by washing with HEPES-buffered HBSS. The DCF fluorescence intensity was measured via flow cytometry, recording 10,000 events for each analysis. Data were analyzed using Kaluza Analysis Software version 2.1 (Beckman Coulter) [59 (link)].
8
Membrane Labeling and Cell Sorting
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For membrane labeling, TILs and cancer cells were stained with fluorochrome-coupled mAbs incubated for 20 minutes at 4°C, protected from light, and then washed with 1× PBS. Intracellular staining was performed after permeabilization with the FoxP3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, catalog 00-5523-00) and intracellularly labeled with anti-Foxp3 mAb (eBiosciences, clone PCH101) and anti-Ki67 (BD Biosciences, catalog 556027), following the manufacturer’s protocol. Cell samples were acquired on a BD LSRFortessa X-20 flow cytometer (BD Biosciences) with single-stained Ab-capturing beads used for compensation (CompBeads, BD Biosciences, catalog 552843). For cell sorting, Zombie Aqua– cells and anti-HLA-I+ and HLA-I– cells after BCG coincubation for 24 hours were sorted on a BD FACSAria III Fusion (BD Biosciences). Data were analyzed with Kaluza Analysis software, version 2.1 (Beckman Coulter). MB49 and UPPL1541 cell lines were cultured in vitro into media with or without recombinant mouse IFN-γ (BioLegend, catalog 575306) for 24 hours and subsequently stained for HLA-I with FITC anti-mouse H-2Kb Ab (BioLegend, catalog 1165005) and run by flow cytometry.
9
Comprehensive Immune Profiling by Flow Cytometry
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Using flow cytometry, blood samples were processed with an extensive panel of human-specific monoclonal antibodies. These antibodies included anti-CD3 PerCp, anti-CD4 BV421, anti-CD8 BV605, anti-CD19 FITC, and anti-CD45 AF700, along with other antibodies targeting immune checkpoints and pertinent markers, such as anti-PD-1 APC, anti-PD-L1 PE, anti-CTLA-4 PE, anti-CD86 APC, anti-CD200 PE, and anti-CD200R APC. All of these antibodies were sourced from BioLegend (San Diego, CA, USA). For accurate gating during cytometric analysis, FMO controls were incorporated specifically for the immune checkpoints’ antibodies.
After the antibody incubation stage, red blood cells were lysed using a specific buffer from BD (Franklin Lakes, NJ, USA). This lysing solution was prepared following the manufacturer’s protocol to ensure optimal cell lysis. The post-lysis cell suspension was then washed and analyzed on the CytoFLEX LX cytometer (Beckman Coulter, Indianapolis, IN, USA). For subsequent data interpretation, Kaluza Analysis software, version 2.1, also from Beckman Coulter, was used. The CytoFLEX LX system’s consistent quality was upheld using CytoFLEX Ready to Use Daily QC Fluorospheres reagents (Beckman Coulter, Indianapolis, IN, USA).Figure 1 and Figure 2 present the results of the sample analysis.
After the antibody incubation stage, red blood cells were lysed using a specific buffer from BD (Franklin Lakes, NJ, USA). This lysing solution was prepared following the manufacturer’s protocol to ensure optimal cell lysis. The post-lysis cell suspension was then washed and analyzed on the CytoFLEX LX cytometer (Beckman Coulter, Indianapolis, IN, USA). For subsequent data interpretation, Kaluza Analysis software, version 2.1, also from Beckman Coulter, was used. The CytoFLEX LX system’s consistent quality was upheld using CytoFLEX Ready to Use Daily QC Fluorospheres reagents (Beckman Coulter, Indianapolis, IN, USA).
10
Multivariate Flow Cytometry Analysis
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Flow cytometry data were analyzed with Kaluza Analysis Software version 2.1 (Beckman Coulter). Populations of interest were manually gated and JMP 14.2.0 (SAS Institute) was used for uni/multivariate statistical analysis. Raw or normalized Mean Fluorescent Intensity (MFI) values were considered depending on the condition studied. Raw MFIs were used to study basal marker expression levels while LPS or LPS+IFX conditions were normalized by the negative or LPS conditions respectively, thereby obtaining stimulation indexes (Stim. Index). Unsupervised analysis using JMP’s response screening platform was used to identify most discriminative parameters based on t-student or ANOVA test results and false discovery rate (FDR) corrected p-values. Principal component analyses (PCA) were carried out considering most discriminative features. To further study divergences between cohorts, box plot representations were used and hierarchical clustering was conducted. Differences in MFI and/or stimulation indexes were analyzed by a non-parametric Wilcoxon test for which *p-values inferior to 0.05 were considered significant.
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