Journal: Frontiers in Immunology

Article Title: Distinct Signatures in the Receptor Repertoire Discriminate CD56bright and CD56dim Natural Killer Cells

doi: 10.3389/fimmu.2020.568927

Figure Lengend Snippet: Overview of workflow and conducted experiments. (A) Primary human NK cells from peripheral blood of healthy donors were enriched by density gradient centrifugation and subsequent immunomagnetic negative selection. Enriched cells were cultured overnight in low-dose IL-2 (50 U/ml) and IL-15 (5 ng/ml). (B) For flow cytometric identification and purity assessment of NK cells, cells were stained for the expression of the lineage markers CD3, CD14, CD19, CD16, and CD56. For further discrimination of educated and uneducated sub-populations, NK cells were additionally labeled with antibodies against NKG2A, KIR3DL1, KIR2DL1, and KIR2DL2/L3. NK cells from up to four individual donors were subsequently multiplexed for further NK-cell phenotyping using α-hCD45. Multiplexed NK cells were stained with 384 antibodies and controls against surface antigens (n = 18). (C) Controls and surface antigens used for identification of NK cells and their subsets were excluded from further analysis. Therefore, expression of 338 surface antigens was assessed on NK cells. Exemplary dot plots show differential patterns of expression. (D) Downstream analyses focused on phenotypic characterization of NK cells and assessment of inter-individual variability in surface expression as well as phenotypic differences between CD56bright and CD56dim NK cells ( – ).

Article Snippet: Primary NK cells were enriched from PBMCs using an immunomagnetic negative selection strategy (EasySep Human NK cell Isolation Kit, Stemcell Technologies, VA, Canada) according to the manufacturer’s protocol.

Techniques: Gradient Centrifugation, Selection, Cell Culture, Staining, Expressing, Labeling

Journal: Frontiers in Immunology

Article Title: Distinct Signatures in the Receptor Repertoire Discriminate CD56bright and CD56dim Natural Killer Cells

doi: 10.3389/fimmu.2020.568927

Figure Lengend Snippet: Phenotypic characterization of bulk NK cells and assessment of inter-individual variability. Surface expression of 338 different surface molecules on NK cells was assessed using flow cytometry (n = 18). (A) Distribution of expression of surface antigens on NK cells displayed as % positive NK cells. Bar graphs show IQR of expression of the respective surface molecule and black bars indicate the median. Surface molecules were ranked descending by median expression. (B) Summary table shows numeric results for overall surface expression. A total of 93 surface molecules exhibited a median expression of >5% of NK cells. (C) Venn diagram displaying the number of markers identified on NK cells with a median expression of >5% and/or a minimum inter-donor range of ≥5 p.p. (D) Principal component analysis was performed on all 136 surface antigens with a minimum inter-donor range of ≥5 p.p. (i.e. on all molecules with inter-individual variability in surface expression). Circle size indicates quality of representation (cos2) of each surface molecule.

Article Snippet: Primary NK cells were enriched from PBMCs using an immunomagnetic negative selection strategy (EasySep Human NK cell Isolation Kit, Stemcell Technologies, VA, Canada) according to the manufacturer’s protocol.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Distinct Signatures in the Receptor Repertoire Discriminate CD56bright and CD56dim Natural Killer Cells

doi: 10.3389/fimmu.2020.568927

Figure Lengend Snippet: Intra-individual differences in surface expression between CD56bright and CD56dim NK-cell subsets. Expression of 338 different surface antigens on CD56bright and CD56dim NK cells was assessed using flow cytometry (n = 18). (A) Overall distribution of surface expression on CD56bright and CD56dim NK-cell subsets displayed as % positive NK cells. Bar graphs show IQR of expression of the respective molecule and white/black bars indicate the median. Surface antigens were ranked descending by median expression for each subset separately, thus their orders are not identical. (B) Summary tables show numeric results for overall surface expression on CD56bright and CD56dim NK cells. Eighty-four and 92 surface molecules were expressed on CD56bright and CD56dim NK cells, respectively (>5% median expression). A total of 104 surface antigens were consistently expressed on at least one of these NK-cell subsets. (C) Expression of all 338 surface antigens was compared between CD56bright and CD56dim NK cells. Wilcoxon signed rank tests were performed between subpopulations with an FDR adjustment for test multiplicity. Data was analyzed regarding median differences in expression >5 p.p. and statistical significance (FDR-adjusted p < 0.05). Representative bar graphs show exemplary data for all possible combinations of the two criteria. (D) Summary table shows numeric results. All 70 surface molecules with statistically significant differences >5 p.p. in expression between CD56bright and CD56dim NK cells were grouped according to their function.

Article Snippet: Primary NK cells were enriched from PBMCs using an immunomagnetic negative selection strategy (EasySep Human NK cell Isolation Kit, Stemcell Technologies, VA, Canada) according to the manufacturer’s protocol.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Distinct Signatures in the Receptor Repertoire Discriminate CD56bright and CD56dim Natural Killer Cells

doi: 10.3389/fimmu.2020.568927

Figure Lengend Snippet: Receptor expression-based unsupervised hierarchical clustering of CD56bright and CD56dim NK cells. NK cells were assessed for surface expression of 338 different surface antigens using flow cytometry (n = 18). Wilcoxon signed rank tests were performed between subpopulations with a Benjamini and Hochberg FDR adjustment for test multiplicity. Seventy molecules displayed a median difference >5 p.p. between CD56bright and CD56dim NK cells and were statistically significant (FDR-adjusted p < 0.05). Heatmap displaying frequency of surface antigen-expressing NK cells based on these 70 molecules in CD56bright (left) and CD56dim NK cells (right) for each donor. Field color indicates median surface expression (in %) on the respective NK-cell subset. Black fields represent missing or excluded data (i.e. due to insufficient cell population size <100 cells). Unsupervised hierarchical clustering confirmed the major phenotypic differences as data clearly clustered into CD56bright and CD56dim NK cells based on expression of surface molecules.

Article Snippet: Primary NK cells were enriched from PBMCs using an immunomagnetic negative selection strategy (EasySep Human NK cell Isolation Kit, Stemcell Technologies, VA, Canada) according to the manufacturer’s protocol.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Distinct Signatures in the Receptor Repertoire Discriminate CD56bright and CD56dim Natural Killer Cells

doi: 10.3389/fimmu.2020.568927

Figure Lengend Snippet: Intra-individual differences in surface expression of adhesion molecules and NK-cell receptors between CD56bright and CD56dim NK-cell subsets. NK cells were assessed for surface expression of 338 different surface antigens using flow cytometry (n = 18). Wilcoxon signed rank tests were performed between subpopulations with a Benjamini and Hochberg FDR adjustment for test multiplicity. Seventy molecules displayed a median difference >5 percentage points between CD56bright and CD56dim NK cells and were statistically significant (FDR-adjusted p < 0.05). Those 70 surface molecules were subsequently grouped according to their function. Bars indicate median expression (% positive NK cells) of all members of the groups titled adhesion molecules (A) and NK-cell receptors (B) .

Article Snippet: Primary NK cells were enriched from PBMCs using an immunomagnetic negative selection strategy (EasySep Human NK cell Isolation Kit, Stemcell Technologies, VA, Canada) according to the manufacturer’s protocol.

Techniques: Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Distinct Signatures in the Receptor Repertoire Discriminate CD56bright and CD56dim Natural Killer Cells

doi: 10.3389/fimmu.2020.568927

Figure Lengend Snippet: Intra-individual differences in surface expression of cytokine and chemokine receptors, activation and differentiation, survival/apoptosis, and miscellaneous molecules between CD56bright and CD56dim NK-cell subsets. NK cells were assessed for surface expression of 338 different surface antigens using flow cytometry (n = 18). Wilcoxon signed rank tests were performed between subpopulations with a Benjamini and Hochberg FDR adjustment for test multiplicity. Seventy molecules displayed a median difference >5 percentage points between CD56bright and CD56dim NK cells and were statistically significant (FDR-adjusted p < 0.05). Those 70 surface molecules were subsequently grouped according to their function. Bars indicate median expression (% positive NK cells) of all members of the groups titled cytokine and chemokine receptors (A) , activation and differentiation (B) , survival/apoptosis (C) , and miscellaneous (D) .

Article Snippet: Primary NK cells were enriched from PBMCs using an immunomagnetic negative selection strategy (EasySep Human NK cell Isolation Kit, Stemcell Technologies, VA, Canada) according to the manufacturer’s protocol.

Techniques: Expressing, Activation Assay, Flow Cytometry

Journal: Bio-protocol

Article Title: In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation

doi: 10.21769/BioProtoc.2386

Figure Lengend Snippet: A. Human neutrophils were isolated from fresh peripheral blood of healthy donors by gradient centrifugation (step A1). The neutrophil layer at the interface of H1077 and H1119 were collected. PBMC, peripheral blood mononuclear cells; PMN, polymorphonuclear leukocytes; RBC, red blood cells. B. The cell viability was analyzed with PI dye and the purity of the viable neutrophils was analyzed using a human CD15 antibody within the PI negative gate. C. Human neutrophils were isolated from buffy coat with the MACSxpress method (step A2). The cell viability was analyzed with PI dye and the purity of the viable neutrophils was analyzed with human CD15 antibody within the PI negative gate. D. Murine bone marrow neutrophils were isolated by magnetic sorting using a negative depletion method (step A3). The cell viability was analyzed with PI dye and the purity of the viable neutrophils was analyzed with a mouse Ly6G (Gr-1) antibody within the PI negative gate. Gray histograms: PI dye or CD15 antibody or Gr-1 antibody; open histogram: unstained control or isotype antibody.

Article Snippet: Isolate neutrophils by immunomagnetic separation (MACS) using the negative depletion method with mouse neutrophil isolation kit (Miltenyi), following the manufacturer’s instructions.

Techniques: Isolation, Gradient Centrifugation, Control

Journal: Bio-protocol

Article Title: In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation

doi: 10.21769/BioProtoc.2386

Figure Lengend Snippet: 2 x 105 human neutrophils (A) or murine neutrophils (B) were activated by 100 ng/ml PMA for 3 h in wells of glass chamber slide. Resting neutrophils with DMSO served as negative control. Slides were fixed and labelled with anti-DNA/Histone H1 antibody followed by AF555-conjugated anti-mouse IgG (red) and nuclei were counterstained with DAPI (blue) (steps B1 and B2). C. Immunostaining of human neutrophil marker CD15 (green) and NET marker DNA/Histone H1 (red) on paraffin-embedded skin section from human vasculitis patients. D. Immunostaining of the murine neutrophil marker Ly6G (green) and NET marker DNA/Histone H1 (red) on paraffin-embedded skin section from mice with induced immune complex-mediated vasculitis (step B3). Scale bars = 100 µm.

Article Snippet: Isolate neutrophils by immunomagnetic separation (MACS) using the negative depletion method with mouse neutrophil isolation kit (Miltenyi), following the manufacturer’s instructions.

Techniques: Negative Control, Immunostaining, Marker

Journal: PLoS ONE

Article Title: Targeting Tumour-Initiating Cells with TRAIL Based Combination Therapy Ensures Complete and Lasting Eradication of Multiple Myeloma Tumours In Vivo

doi: 10.1371/journal.pone.0035830

Figure Lengend Snippet: The RPMI8226, NCI H929 and OPM2 cells (1×10 5 /ml) were grown for 18 hours and then left untreated or treated with TRAIL (100 ng/ml) (3A) or DOX (500 ng/ml) (3B) for 6 hours. Cell death was assessed by annexin V staining and cellular uptake of either 7AAD for TRAIL treatment (Fig. 3A) or TOPRO3 for DOX treatment (Fig. 3B). In vitro apoptosis of myeloma cells was assessed by flow cytometric analysis of annexin V-APC/7 AAD stained cells (TRAIL treated) or annexin V-FITC/TOPRO3 stained cells (DOX treated).

Article Snippet: The human MM cell lines, RPMI8226 and NCI H929 were obtained from the American Type Culture Collection (Rockville MD, USA) and the OPM-2 line from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Staining, In Vitro

Journal: PLoS ONE

Article Title: Targeting Tumour-Initiating Cells with TRAIL Based Combination Therapy Ensures Complete and Lasting Eradication of Multiple Myeloma Tumours In Vivo

doi: 10.1371/journal.pone.0035830

Figure Lengend Snippet: After immunomagnetic separation, CD138 + and CD138 − RPMI8226 (A), NCI H929 (B) and OPM2 (C) cells (1×10 5 /ml) were incubated with DOX (500 ng/ml) and TRAIL (25 ng/ml) alone for 24 h, or TRAIL for 24 h after initial pre-incubation with DOX (500 ng/ml) for 24 h. Cytotoxicity was measured by MTS assay and expressed as a percentage of the untreated control sample. For details see . Data represent the mean ±1SD of the three independent experiments.* = p<0.05

Article Snippet: The human MM cell lines, RPMI8226 and NCI H929 were obtained from the American Type Culture Collection (Rockville MD, USA) and the OPM-2 line from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Immunomagnetic Separation, Incubation, MTS Assay, Control

Journal: PLoS ONE

Article Title: Targeting Tumour-Initiating Cells with TRAIL Based Combination Therapy Ensures Complete and Lasting Eradication of Multiple Myeloma Tumours In Vivo

doi: 10.1371/journal.pone.0035830

Figure Lengend Snippet: Purified CD138 + and CD138 − cells were incubated with mouse anti-human TRAIL receptor R1 and R2 PE-conjugated monoclonal antibodies and analysed by flow cytometry to determine the percentage of labelled cells. The numbers indicate the percentage of receptor-positive cells. A, Representative histograms showing expression of R1 (a and b, thick solid lines) and R2 (c and d, thick solid lines) in CD138 + (a and c) and CD138 − RPMI8226 cells (b and d). The thin lines represent the appropriate isotype controls. B and C, Numerical representation of the percentage of CD138 + and CD138 − NCI H929 and OPM2 cells expressing R1 and R2 receptors.

Article Snippet: The human MM cell lines, RPMI8226 and NCI H929 were obtained from the American Type Culture Collection (Rockville MD, USA) and the OPM-2 line from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Purification, Incubation, Bioprocessing, Flow Cytometry, Expressing

Journal: Oncology letters

Article Title: Selective surface marker and miRNA profiles of CD34 + blast-derived microvesicles in chronic myelogenous leukemia.

doi: 10.3892/ol.2017.6336

Figure Lengend Snippet: Figure 1. CD123 expression is significantly increased in CD34+ cells of patients with CP‑CML and BC‑CML, compared with healthy donors. The gating strategy applied to determine the proportion of CD123+ cells in CD34+ subsets was: (A) P1, active cells; (B) isotype control; (C) Q2‑2, CD34+ blasts; (D) isotype control; (E) Q2‑8, CD34+ CD123+ blasts. (F) Proportion of CD123+ cells within CD34+ populations from healthy donors, and patients with CP‑CML and BC‑CML determined using multicolor flow cytometry. Each dot represents an individual tissue sample. P=0.105, N vs. CP‑CML; **P<0.01, CP‑CML vs. BC‑CML. CD, cluster of differentiation; CP, chronic‑phase; CML, chronic myelogenous leukemia; BC, blast crisis; N, healthy donors; SSC, side scatter; FSC, forward scatter; PE, phycoerythrin; Cy7, cyanine 7; Q, quadrant; Ig, immunoglobulin.

Article Snippet: CD34+ cells (>92% pure) were isolated using immunomagnetic separation by positive selection of cells (human CD34 microbead kit; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's protocol.

Techniques: Expressing, Control, Flow Cytometry

Journal: Oncology letters

Article Title: Selective surface marker and miRNA profiles of CD34 + blast-derived microvesicles in chronic myelogenous leukemia.

doi: 10.3892/ol.2017.6336

Figure Lengend Snippet: Figure 2. CD123 expression is significantly increased in CD34+ blast‑derived MVs of patients with CP‑CML and BC‑CML compared with healthy donors. Flow cytometric analysis and representative cytograms of CD123+CD34+ blast‑derived MVs in patients with CML. (A) Fluorosphere beads of a known size were used as a standard to set the MV gate. P3 represents 1 µm beads. (B) P1 represents 3 µm beads. (C) P1 and P3 represent 3 µm and 1 µm beads, respectively. (D) Based on the calibrating beads above, P2 represents the MV gate. (E) IgG1‑FITC was set as the negative control for calcein‑AM+ MVs. (F) Events in the MV gate (P4) were analyzed to differentiate calcein‑AM+ MVs from the background signal. (G) Q2‑3, calcein‑AM+CD34+ MVs; (H) CD123 isotype control, Q2‑2, calcein‑AM+CD34+CD123‑ MVs. (I) Q2‑2, calcein‑AM+CD34+CD123+ MVs. (J) The proportion of CD123+ MV within calcein‑AM+CD34+ populations from N‑MV and patients with C‑MV and B‑MV was determined using multicolor flow cytometry. P=0.226, N‑MV vs. C‑MV; **P<0.01, C‑MV vs. B‑MV. CD, cluster of differentiation; CP, chronic‑phase; CML, chronic myelogenous leukemia; BC, blast crisis; MV, microvesicle; N‑MV, healthy; C‑MV, chronic‑phase CML; B‑MV, blast crisis CML; SSC, side scatter; FSC, forward scatter; PE, phycoerythrin; Cy7, cyanine 7; Q, quadrant; Ig, immunoglobulin; FITC, fluores cein isothiocyanate; Cal‑AM, calcein acetoxymethyl ester.

Article Snippet: CD34+ cells (>92% pure) were isolated using immunomagnetic separation by positive selection of cells (human CD34 microbead kit; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's protocol.

Techniques: Expressing, Negative Control, Control, Flow Cytometry

Journal: Oncology letters

Article Title: Selective surface marker and miRNA profiles of CD34 + blast-derived microvesicles in chronic myelogenous leukemia.

doi: 10.3892/ol.2017.6336

Figure Lengend Snippet: Figure 5. Validation of miRNA expression levels using the reverse transcription‑quantitative polymerase chain reaction. Expression levels of miR‑627‑5p, miR‑483‑5p, miR‑638 and miR‑1290 in an independent set of primary CD34+ blasts and blast‑derived MVs were determined. miRNA expression in blasts was set as 1; the relative expression of hsa‑miR‑627‑5p, hsa‑miR‑483‑5p, hsa‑miR‑638 and hsa‑miR‑1290 in MVs was 2.09±0.35, 2.14±0.01, 5.59±0.69 and 3.92±0.46, respectively. Results are presented as the mean ± standard deviation. *P<0.05. miRNA, microRNA; CD, cluster of differentiation. MV, microvesicle; hsa, human.

Article Snippet: CD34+ cells (>92% pure) were isolated using immunomagnetic separation by positive selection of cells (human CD34 microbead kit; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's protocol.

Techniques: Biomarker Discovery, Expressing, Polymerase Chain Reaction, Standard Deviation

Journal: Channels (Austin, Tex.)

Article Title: Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells.

doi: 10.1080/19336950.2020.1859753

Figure Lengend Snippet: Figure 2. Comparison of viability and purity after the different separation techniques. Viability was determined with 7-AAD staining while purity was expressed as a percentage of CD4+ cells in the living cell population (see Methods). Measurements were made via flow cytometry, subsequently after the separation (values are plotted as mean±SD for the number of independent experiments indicated in the bar). No significant differences were found among the groups for either viability or purity (one-way analysis of variance, p > 0.05). REAlease®: measured after the complete removal of bead-antibody and REAlease® complex, in label-free configuration (see Figure 1), Negative selection: immunomagnetic negative selection, Sorted: positive selection by flow cytometry.

Article Snippet: Immunomagnetic negative selection-based separation (CD4+ T Cell Isolation Kit Cat. No.: 130–096-533 Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany) was prepared to isolate cells without labeling of the CD4 molecule, and these cells served as the “Control” configuration (Figure 1).

Techniques: Comparison, Staining, Flow Cytometry, Selection