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14 protocols using version 9

1

Apoptosis Detection by Annexin V-FITC

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The Annexin V-FITC staining kit was used to identify apoptotic cells. Cells were treated with fisetin for 48 hours and then with cabazitaxel for 24 hours, washed, centrifuged and the cell-pellet suspended in Annexin V-FITC binding buffer. Cell suspensions were then incubated with Annexin V-FITC conjugate and Propidium iodide solution. Data were collected on a Becton-Dickinson FACSCalibur (San Jose, CA) and analyzed in FlowJo, Version 9.7 (FLowJo, LLC, Ashland, OR).
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Early Apoptosis Detection in Colon Cancer

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The Annexin V-FITC early apoptosis detection kit (Cell Signaling Technology, Danvers, MA) was used to identify apoptotic cells within a cell population. The human colon cancer cells were grown to ~70% confluence and treated with fisetin (30–90 μM), 5-FU (50 μM) or their combination for 48 hr. The cells were washed, centrifuged and the cell pellet was resuspended in Annexin-V-FLUOS labeling solution (prepared by prediluting Annexin-V-FLUOS labeling reagent in incubation buffer and adding propidium iodide solution). The data was collected on a Becton–Dickinson FACSCalibur (San Jose, CA) and analyzed in FlowJo, Version 9.7 (FLowJo, LLC, Ashland, OR).
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3

Flow Cytometry Analysis of Immune Cells

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Flow cytometry data were analyzed using FlowJo Version 9.6. Results from single-stained and unstained mouse kappa beads were used to calculate compensations. Cell doublets were excluded using forward scatter-area versus forward scatter-height parameters. T cells, NK cells, neutrophils and epithelial cells (for explant samples) were also excluded from the analyses. The MFI for HLA-DR (explants) and CD40 (whole blood) were determined by FlowJo, and samples were excluded if any event numbers for cell populations were less than 20 (for activation only). Any sample with less than 50,000 events during acquisition was also excluded from the analyses. GraphPad Prism Version 6 was used for data presentation and statistical analysis. Comparisons within the same sample were done using a Wilcoxon matched-pairs signed rank test. Comparisons between the different samples were assessed by Mann-Whitney U test, and p values less than 0.05 were considered statistically significant.
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4

Identification of CD8+ T-cell Epitopes

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Lymphocytes were obtained from the pneumonic lung by bronchoalveolar lavage (BAL) and adherent cells were removed by incubating on plastic for 1 h at 37 °C. Single-cell preparations of spleen were enriched for CD8 T cells by panning on tissue culture plates coated previously with AffiniPure goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch Labs). CD8+ T-cell responses to each candidate epitope were tested via intracellular cytokine staining. Enriched lymphocytes from spleens or bronchoalveolar lavage of mice infected 10 days previously with IAV were incubated with 10 U/ml IL-2 (Roche) and 1 μg/ml Golgi-plug (BD Biosciences) in the presence or absence of 1 μM synthetic peptide in 96-well plates and cultured for 5 h at 37 °C and 5% CO2. Cells were then stained for surface expression of CD8α and intracellular IFN-γ and TNF. Data was acquired by flow cytometry (FACSCanto, BD Biosciences) and analysis was performed using Flowjo version 9.6 (FlowJo LLC, Ashland). Single viable live CD8+ lymphocytes were gated for analysis (Supplementary Fig. 7). A response was deemed positive if the test response (with background subtracted) was equal to or greater than 2× the average background values.
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5

Intracellular Cytokine Staining Assay

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Cells were surface stained for CD8 and CD3, and then stained intracellularly for IFNg, IL-2, and TNF, using the Cytofix/Cytoperm kit (BD Pharmingen). Data were analyzed using FlowJo, version 9.6 (FlowJo LLC, Ashland, OR), Pestle, version 1.8, and SPICE, version 5.3, software (Mario Roederer, National Institutes of Health, Bethesda, MD) .
For analyses of cytokine production by in vitro stimulated cells, GolgiPlug was added 5 hr prior to harvest, and then cells were stained as described above.
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6

Multiparametric Flow Cytometry Analysis

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Batched stored samples were thawed quickly at 37°C and washed twice with 1X BD PermWash buffer. Cells were then incubated in 1X BD PermWash for 10 min before incubation with the antibody cocktail mix in 2% FCS at 4°C for 45 min. After incubation, cells were washed twice with PBS (2% FCS) and the resuspended in 0.3 mL PBS for cell acquisition using a Beckton Dickinson LSRII flow cytometer (SORF model). The following monoclonal antibody-fluorochrome conjugates were used: IL-2-R-phycoerythrin (PE), CD8-V500, IFN-γ-Alexa Fluor-700, TNF-α-PE-Cy7, Ki67-Fluorescein isothiocyanate (FITC), all from BD, CD27 PE-Cy5, HLA-DR- Allophycocyanin-Cy7 (APC-Cy7), CD3-BV650 (Biolegend), CD4 PE-Cy5.5 (Invitrogen), CD45RA PE-Texas Red-X (Beckman Coulter). A minimum of 50,000 ViViD negative (viable) CD3 events were collected using FACS DIVA v6 software. Post-acquisition compensation and analysis was performed in FlowJo version 9 (FlowJo, LLC). Supplementary Figure 1 shows the gating strategy employed.
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7

Multiparametric Flow Cytometry for T Cell Polyfunctionality

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Staining and flow cytometry were conducted as previously described (34 (link)). In brief, batched stored samples were thawed quickly and washed, incubated in BD PermWash, and then stained with the antibody cocktail mix. After incubation, cells were washed and then resuspended in PBS for cell acquisition using a BD LSRII flow cytometer (SORF model). The following monoclonal antibody–fluorochrome conjugates were used: IL-2-R–phycoerythrin (PE), CD8-V500, IFN-γ–Alexa Fluor 700, TNF-α–PE-Cy7, Ki67–fluorescein isothiocyanate (FITC), all from BD; CD27–PE-Cy5, HLA-DR–allophycocyanin-Cy7 (APC-Cy7), CD3-BV650 (BioLegend); CD4–PE-Cy5.5 (Invitrogen); and CD45RA–PE-Texas Red-X (Beckman Coulter). A minimum of 50,000 ViViD-negative (viable) CD3+ events were collected using BD FACSDiva v6 software. Postacquisition compensation and analysis was performed in FlowJo version 9 (FlowJo, LLC). Supplemental Figure 2 shows the gating strategy employed. Measurable response to BCG was characterized by the polyfunctionality of the CD3+CD4+ T cell response assessed by analyzing permutations of TNF-α, IL-2, and IFN-γ expression after stimulation using combinatorial polyfunctionality analysis of antigen-specific T cell subsets (COMPASS) (35 (link)). PFS and FS, which summarize the functional profile of each subject, were calculated from posterior probabilities as described previously (35 (link)).
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8

Evaluating CRT Modulation on HL-60 Cells

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To evaluate the effect of CRT siRNA transfection and CRT overexpression on HL-60 cells, CD11b and CD33 levels were evaluated. Cells from the different groups (with untreated HL-60 cells included as the normal control) were collected, centrifuged at 500 × g for 5 min, washed twice with PBS, and the cell concentration was adjusted to 1×106 cells/ml. Cells were then fixed with 70% ethanol. Prior to FCM analysis, the cells were washed twice again with PBS and 500 µl 0.2% Triton-X 100 was added. The mixture was incubated on ice for 10 min. After washing the samples twice again with PBS, 50 µl primary antibody was added and cells were incubated on ice for 30–45 min. After washing with PBS, 50 µl specific fluorescent secondary antibody was added, and cells were incubated on ice for 30 min. Subsequent to washing with PBS, the cells were immediately measured by FCM (FlowJo version 9; FlowJo, LLC, Ashland, OR, USA).
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9

Murine Hematopoietic Engraftment Analysis

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Blood was collected from the retro-orbital sinus, facial vein, or tail vein of anesthetized mice to assess engraftment of human cells. Blood was lysed with the BD FACS Lyse Wash Assistant (BD Biosciences, San Jose, CA). Cells were stained with antibodies to human CD45 (BD Biosciences, # 561864, APC), mouse CD45 (BD Biosciences, # 559864, PE). Samples were assayed on the BD LSR II or LSR Fortessa (BD Biosciences) and data were analyzed with FlowJo version 9 (FlowJo, LLC, Ashland, OR). Samples were sorted for hCD45 and YFP on the BD Aria (BD Biosciences).
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10

Flow Cytometry for ABCG2 Expression

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Expression levels of ABCG2 were assessed by flow cytometry as previously described91 (link), using PE-conjugated anti-ABCG2 antibody (R&D Systems, Minneapolis, MN). Results were analyzed relative to negative isotype control, PE-conjugated IgG2b (DakoCytomation, Denmark A/S) with an LSRFortessa™ X-20 and FlowJo version 9 (FlowJo LLC, Ashland, OR).
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