The following monoclonal antibodies (MAbs) were used for phenotypic analysis: (i) anti-CD27-APC-eFluor780, anti-CD45RA-phycoerythrin (PE), and anti-CD127-eFluor450 (eBioscience, San Diego, CA); (ii) anti-CD3-Pacific Blue, anti-CD8-AmCyan, anti-CD8-V500, anti-CD11a-fluorescein isothiocyanate (FITC), anti-CD95-PE, anti-Ki67-FITC, and anti-CCR7-PE-Cy7 (BD Biosciences, Heidelberg, Germany); (iii) anti-CCR7-FITC (R&D Systems); and (iv) anti-PD-1-PE-Cy7 (BioLegend, San Diego, CA). 7-Aminoactinomycin D (7-AAD; Viaprobe; BD Biosciences) was used for the exclusion of dead cells. All samples were acquired using a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., Ashland, OR).
Anti ccr7 fitc
Manufactured by R&D Systems
Sourced in Germany
Anti-CCR7 FITC is a fluorochrome-conjugated antibody that recognizes the CCR7 chemokine receptor. CCR7 is involved in the migration and homing of lymphocytes. The Anti-CCR7 FITC antibody can be used to detect and quantify CCR7-expressing cells by flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd27 apc efluor780, by Thermo Fisher Scientific (2 mentions)
Anti cd8 amcyan, by BD (2 mentions)
Anti cd4 apc cy7, by BD (2 mentions)
Anti cd3 bv421, by BD (2 mentions)
Anti cd34 fitc, by BD (2 mentions)
Anti cd25 pe cy7, by BD (2 mentions)
Anti ccr6 pe, by BD (2 mentions)
Anti cd27 pe, by BD (2 mentions)
Apc anti cd10, by BD (2 mentions)
Anti igg apc, by BD (2 mentions)
Anti il 22 pe, by Thermo Fisher Scientific (2 mentions)
Anti cd27 pe cy7, by BD (2 mentions)
Anti il 2 bv711, by BD (2 mentions)
Anti il 17f bv786, by BD (2 mentions)
Anti tnf α buv395, by BD (2 mentions)
Anti il 13 bv421, by BD (2 mentions)
Anti il 17a apccy7, by BioLegend (2 mentions)
Anti ifn γ bv605, by BD (2 mentions)
Anti cd19 bv711, by BD (2 mentions)
Anti cd127 bv650, by BioLegend (2 mentions)
8 protocols using anti ccr7 fitc
1
Phenotypic Characterization of Immune Cells
2
CLL B Cell Surface Marker Analysis
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Freshly isolated CLL B cells were cultured with and without MSCs and treated with 5μM kinase inhibitors as above described. Cells (5×105 for each assay) were collected after 48h, leaving intact the adherent layer, and stained with anti-CD49d PE (BD Biosciences), anti-CCR7 FITC (R&D Systems Inc., Minneapolis, MN, USA), anti-CXCR4 PE (R&D Systems Inc.) anti-CXCR3 PE-Cy5.5 (BD Biosciences, Franklin Lakes, NJ, USA), anti-CXCR5 FITC (R&D Systems Inc.), and anti-CD19 APC (BD Biosciences) monoclonal antibodies. Cells were washed with PBS1X and incubated with saturating concentrations of the appropriate antibodies for 15 minutes at room temperature. 20,000 total events were acquired using FACSCanto A (Becton Dickinson) and the data were analysed by FACSDiva 7 software. Samples were gated on intact cells by forward scatter (FSC) vs side scatter (SSC). For analysis, a second gating step on CD19+ cells was used. Here, we used a difference between the Mean Fluorescence Intensity (MFI) of fully-stained samples and the Fluorescence Minus One (FMO) controls.
3
Comprehensive Immune Cell Profiling
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The following mAbs were used: anti-CD20 BUV395, anti-CD10 APC, anti-CD4 BUV737, anti-CD4 APCCy7, anti-CD25 PECy7, anti-CD27 PECy7, anti-CD27 PE, anti-CD45RA BV605, anti-CXCR5 A647, anti-IgG APC, anti-IgG PE, anti-IgG BV605, anti-CD8 BUV395, anti-PD1 BV605, Streptavidin-PerCPCy5.5, anti-IgM PerCPCy5.5, anti-CD3 BV421, anti-IL-2 BV711, anti-IL-9 PerCPCy5.5, anti-IL-13 BV421, anti-IL-17F BV786, anti-IFN-γ BV605, anti-TNF-α BUV395, anti-CD19 BV711, anti-CD34 FITC, anti-CCR6 PE (all from Becton Dickinson); anti-CCR7 PECy7, anti-CD127 BV650, anti-IL-17A APCCy7, anti-CD20 Pacific Blue, anti-FoxP3 PE, anti-CXCR3 BV421 (BioLegend); anti-CD45RA PerCPCy5.5 (eBioscience); anti-CCR7 FITC (R&D Systems); anti-IgA-biotin (SouthernBiotech); anti-IL-4 PECy7, anti-IL-21 e660, anti-IL-22 PE (Thermo Fisher Scientific).
4
Macrophage Phenotype Analysis by Flow Cytometry
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At day 2, 4 and 8 the differentiated macrophages were carefully scraped from the CFs or well ground of the culture plates and seeded (5 × 105 cells per well) into 96-well plates. Cells were washed with PBS. Subsequently the Fc receptors were blocked by incubation in a buffer containing 10 µg/ml heat-aggravated human immunoglobulin G (IgG) (Sigma, Deisenhofen, Germany). For extracellular staining the following antibodies were used: anti-CD163-PE (BioLegend, San Diego, CA, USA), anti-CD206-PE, (BD Pharmingen, Heidelberg, Germany) and anti-CCR7-FITC (R&D Systems) and the respective isotype controls to define the unspecific background staining. For intracellular staining cells were fixed, permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization kit (BD Bioscience, Heidelberg, Germany) and anti-CD68-APC (BioLegend) or the respective isotype control was applied. Stained cells were washed three times and resuspended in PBS. Sample acquisition was performed by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany) and geometric mean fluorescence intensities (MFI) were calculated by CellQuest Pro software (Becton Dickinson).
5
Detailed Immunophenotyping of T Cell Subsets
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Immunophenotyping was undertaken following APC-conjugated HLA class I tetramer staining of PBMCs at 37°C for 15 min. Details of the tetramers used can be found in Supplementary Table 5 . Tetramers were conjugated to APC and a true tetramer response was verified through the lack of background staining by gating all CD3+ T cells, against CD8+ T cells and using a tetramer negative control. Surface staining with the following antibodies was then performed: live/dead blue dye (Invitrogen), anti-CD8 Amcyan (BD Biosciences), anti-CD3 APC-Cy7 (Biolegend), anti-PD-1 PercpCy5.5 (BD Biosciences), anti-CTLA4 PE-Cy7, anti-CD244 FITC, and anti-CD160 PE (Biolegend) before washing and flow cytometric analysis. Memory subset analysis utilized the same panel but included anti-CCR7 FITC (R&D systems) and anti-CD45RA AF700 (Biolegend) and omitted anti-CTLA4, anti-CD244, and anti-CD160. Example flow plots can be found in (Figure S1 ).
6
Multiparametric Phenotyping of PBMCs
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A total of 1 x 106 PBMCs were stained with LIVE/Dead fixable violet stain (Invitrogen) for 15 minutes at room temperature (RT), followed by a wash step with phosphate-buffered saline (PBS). Cells were then re-suspended in 50 μL of human serum and incubated with PE-conjugated HLA class II tetramer for 1 h at 37°C and 5% CO2. After three washes with staining buffer (1xPBS supplemented with 0.5% BSA and 2 mM EDTA), the cells were co-stained for 15 minutes at 4°C with saturating amounts of different combinations of the following antibodies: anti-CD3 AmCyan (SK7, BD Biosciences), anti-CD4 PerCPCy5.5 (RPA-T4, eBioscience), anti-CD4 PE-CF594 (RPA-T4, BD Biosciences), anti-CD4 APC-Cy7 (RPA-T4, Biolegend), anti-CD14 Pacific Blue (HCD14, Biolegend), anti-CD19 eFluor450 (H1B19, eBioscience), anti-CD25 APC-Cy7 (BC96, Biolegend), anti-CD27 APC-eFluor780 (O323, eBioscience), anti-CD28 PE-Cy7 (28.2, Biolegend), anti-CD45RA AF700 (H1 100, Biolegend), anti-CD57 APC (HCD56, Biolegend), anti-CD69 PE-Cy7 (FN50, Biolegend), anti-CD127 PerCP-Cy5.5 (HIL-7R-M21, BD), anti-CCR7 FITC (150503, R&D), anti-CX3CR1 PerCP-Cy5.5 (2A9-1, Biolegend), anti-FasL AF488 (14C2, AbD Serotec), anti-NKG2D PE-CF594 (1D11, BD Biosciences), anti-PD-1 PerCP-Cy5.5 (EH12.2H7, Biolegend) and anti-Tim3 APC (F38-2E2, eBioscience). Following a final wash cells were re-suspended in 200 μL of staining buffer for acquisition.
7
Characterization of DC Maturation Markers
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Mature DCs were electroporated without or with IKKβ-RNA as described above and harvested 24 h, 48 h, or 72 h after transfection. Cells were stained at 4°C in FACS buffer for 30 min with the following antibodies: anti-CD40-FITC (BD), anti-CD40-PE (BD), anti-CD25-FITC (Cymbus Technologies, Southampton, Hampshire, United Kingdom or BD), anti-CD25-PE (BD), anti-CD70-PE (BD), anti-OX40L-PE (BD), anti-CD80-FITC (BD), anti-CD83-PE (Miltenyi), anti-CCR7-FITC (R&D Systems), anti-CD86-FITC (Cymbus Technologies), anti-CD86-PE (Miltenyi, BD), and anti-PD-L1-PE (eBioscience) and with matched isotype controls: IgG1-FITC (BD, Miltenyi), IgG1-PE (Miltenyi), IgG2a-FITC (BD), IgG3-PE (eBioscience). The cells were then washed once with FACS buffer and were taken up in FACS buffer or a mixture of equal amounts of FACS-Fix (DPBS with 2% formaldehyde) and FACS buffer. Afterwards, the immunofluorescence was determined using a FACScan cytofluorometer equipped with Cell Quest software (BD). The DCs were discriminated based on their size and granularity by gating in the forward and side scatter channels. The specific mean fluorescence intensities (specific MFI) were calculated by subtraction of the background mean fluorescence intensity obtained with the isotype control antibodies. All values were set in relation to the 24 h control condition to calculate the fold induction.
8
Multiparametric Flow Cytometry of PBMC Subsets
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Cryopreserved PBMCs and their subpopulations were analysed with a 28-color flow cytometry panel, as previously described [34 (link)]. The following mAbs were used: anti-CD20 BUV805, anti-CD10 APC, anti-Vαβ TCR BUV737, anti-CD4 APCCy7, anti-CD25 PECy7, anti-CD27 PECy7, anti-CD27 PE, anti-CD45RA PerCpCy5, anti- CXCR5 BUV615, anti-IgG APC, anti IgG BB660, anti-IgD BV480, anti-IgG BV605, anti-IgA1/A2 PECy5, anti-CD8 BUV496, anti-CD21 BUV563, anti-PD1 BV605, anti-IgM PerCPCy5.5, anti-IgM APC R700, anti-CD3 BV421, anti-IL-2 BV711, anti-IL-9 PerCPCy5.5, anti-IL-13 BV421, anti-IL-17F BV786, anti-IFN-γ BV605, anti-TNF-α BUV395, anti-CD19 BV711, anti-CD34 FITC, anti-CCR6 PE, anti-CD45RA BUV395, anti-CXCR5 BV615 (all from Becton Dickinson); anti-CD20 Pacific Blue, anti-CCR7 PECy7, anti-CD127 BV650, anti-IL-17A APCCy7, anti-CD20 BUV805, anti-CXCR3 BV421, anti-CD3 BV570 (BioLegend); (OptiBuild); anti-CCR7 FITC (R&D Systems); anti-IL-4 PECy7, anti-IL-21 e660, anti-IL-22 PE (Thermo Fisher Scientific).
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