cd112 fitc  (Bio-Rad)

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: AML cells exhibit no, or heterogeneous, expression of DNAM-1 ligands. (A) The indicated AML cell lines were analyzed for expression of CD112, CD155 and CD33 by flow cytometry. Iso refers to immunoglobulin isotype matched control antibody while stain indicates specific antibody staining. (B) The percentage of cells expressing the indicated ligands within populations of the indicated cell type, as analyzed from (A). (C) CD155 expression on the indicated AML cell lines was visualized by confocal microscopy. Scale bar represents 20 µm.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Flow Cytometry, Control, Staining, Confocal Microscopy

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands are required for NK cell activity against AML targets. (A) K562 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (B) K562 cells were FACS sorted as in (A), then used as targets in an NK cell degranulation assay. (C) K562 cells were FACS sorted as in (A), then used as targets in an NK cell chromium release assay. (D) MV4-11 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (E) MV4-11 cells were FACS sorted as in (D), then used as targets in an NK cell degranulation assay. (F) MV-411 cells were FACS sorted as in (D), then used as targets in an NK cell chromium release assay. (G) NK cell chromium release assay against the indicated AML targets (5:1 E:T ratio) in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). (H–I) NK cell chromium release assay at the indicated E:T ratios using FACS sorted high and low CD112/CD155 expressing K562 and MV-411 cells, in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Activity Assay, Staining, Expressing, Degranulation Assay, Release Assay

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands increase the frequency of ‘normal’ NK-target cell synapses. (A–B) FACS sorted (CD112/155 high and low) K562 and MV-411 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later, followed by fixing. The percentage of targets that had conjugated with an NK cell was then quantitated by confocal microscopy. A minimum of 20 fields of view was analyzed and is representative of two independent experiments. (C) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later. After 1 h, cells were fixed, stained with the antibody combinations indicated, then analyzed by confocal microscopy. Representative images of NK-target cell synapses are presented. Scale bar represents 10 µm. (D) The percentage of NK cells that had polarized LFA-1 and perforin to the synapse was quantified from (C). A minimum of 20 NK-target cell synapses was analyzed and data from two independent experiments was pooled. Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Confocal Microscopy, Staining

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: Live imaging reveals that AML cells lacking DNAM-1 ligand expression drive NK cell failed killing. (A) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells labeled with fluo-4 acetoxymethyl AM (green) to indicate calcium signaling, and analyzed by time-lapse microscopy. PtdIns (red) (100 μg/mL) was added to the medium to indicate perforin-induced target membrane puncture. Representative still images at the indicated time-points are depicted (hr:min). (B–C) Individual NK-MV-411 CD112/CD155 high and low contacts were monitored for events that did (successful kill) or did not (failed kill) result in target killing, as indicated by PI influx and apoptotic morphology. (D) The time interval between initial NK-target cell contact and target cell death (PtdIns influx) was analyzed. (E–G) K562 cells were used as targets in the assays described in (B–D) above. All quantification data is pooled from individual movies (n = 3). Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Imaging, Expressing, Labeling, Time-lapse Microscopy, Membrane

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: NK cells preferentially target DNAM-1 ligand-expressing cells and drive clonal selection of DNAM-1 ligand negativity. (A) FACS-sorted K562 CD112/CD155 high and low cells were labeled with CFSE and CTV, respectively, then exposed to NK cells at the indicated E:T ratios. After 4 h, cells were analyzed by flow cytometry and loss of dye was monitored from viable populations. (B) Extended E:T ratio titration for the assay described in (A), using FACS-sorted K562 and MV-411 CD112/CD155 high and low cells as targets. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 2). *p < 0.05 by unpaired Student's t test. (C–D) K562 and MV-411 cells were either exposed to NK cells (1:1 E:T Ratio), or not, for 5 d. Viable AML cells (fixable yellow negative, CD33 positive) were then analyzed for CD112 and CD155 expression by flow cytometry, and compared to parental cells (no NK cell exposure). Bar charts represent the number of CD112/CD155 double positive cells after 5 d in the presence or absence of NK cell exposure. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Selection, Labeling, Flow Cytometry, Titration

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: AML cells exhibit no, or heterogeneous, expression of DNAM-1 ligands. (A) The indicated AML cell lines were analyzed for expression of CD112, CD155 and CD33 by flow cytometry. Iso refers to immunoglobulin isotype matched control antibody while stain indicates specific antibody staining. (B) The percentage of cells expressing the indicated ligands within populations of the indicated cell type, as analyzed from (A). (C) CD155 expression on the indicated AML cell lines was visualized by confocal microscopy. Scale bar represents 20 µm.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Flow Cytometry, Staining, Confocal Microscopy

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands are required for NK cell activity against AML targets. (A) K562 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (B) K562 cells were FACS sorted as in (A), then used as targets in an NK cell degranulation assay. (C) K562 cells were FACS sorted as in (A), then used as targets in an NK cell chromium release assay. (D) MV4-11 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (E) MV4-11 cells were FACS sorted as in (D), then used as targets in an NK cell degranulation assay. (F) MV-411 cells were FACS sorted as in (D), then used as targets in an NK cell chromium release assay. (G) NK cell chromium release assay against the indicated AML targets (5:1 E:T ratio) in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). (H–I) NK cell chromium release assay at the indicated E:T ratios using FACS sorted high and low CD112/CD155 expressing K562 and MV-411 cells, in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Activity Assay, Staining, Expressing, Degranulation Assay, Release Assay

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands increase the frequency of ‘normal’ NK-target cell synapses. (A–B) FACS sorted (CD112/155 high and low) K562 and MV-411 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later, followed by fixing. The percentage of targets that had conjugated with an NK cell was then quantitated by confocal microscopy. A minimum of 20 fields of view was analyzed and is representative of two independent experiments. (C) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later. After 1 h, cells were fixed, stained with the antibody combinations indicated, then analyzed by confocal microscopy. Representative images of NK-target cell synapses are presented. Scale bar represents 10 µm. (D) The percentage of NK cells that had polarized LFA-1 and perforin to the synapse was quantified from (C). A minimum of 20 NK-target cell synapses was analyzed and data from two independent experiments was pooled. Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Confocal Microscopy, Staining

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: Live imaging reveals that AML cells lacking DNAM-1 ligand expression drive NK cell failed killing. (A) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells labeled with fluo-4 acetoxymethyl AM (green) to indicate calcium signaling, and analyzed by time-lapse microscopy. PtdIns (red) (100 μg/mL) was added to the medium to indicate perforin-induced target membrane puncture. Representative still images at the indicated time-points are depicted (hr:min). (B–C) Individual NK-MV-411 CD112/CD155 high and low contacts were monitored for events that did (successful kill) or did not (failed kill) result in target killing, as indicated by PI influx and apoptotic morphology. (D) The time interval between initial NK-target cell contact and target cell death (PtdIns influx) was analyzed. (E–G) K562 cells were used as targets in the assays described in (B–D) above. All quantification data is pooled from individual movies (n = 3). Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Imaging, Expressing, Labeling, Time-lapse Microscopy, Membrane

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: NK cells preferentially target DNAM-1 ligand-expressing cells and drive clonal selection of DNAM-1 ligand negativity. (A) FACS-sorted K562 CD112/CD155 high and low cells were labeled with CFSE and CTV, respectively, then exposed to NK cells at the indicated E:T ratios. After 4 h, cells were analyzed by flow cytometry and loss of dye was monitored from viable populations. (B) Extended E:T ratio titration for the assay described in (A), using FACS-sorted K562 and MV-411 CD112/CD155 high and low cells as targets. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 2). *p < 0.05 by unpaired Student's t test. (C–D) K562 and MV-411 cells were either exposed to NK cells (1:1 E:T Ratio), or not, for 5 d. Viable AML cells (fixable yellow negative, CD33 positive) were then analyzed for CD112 and CD155 expression by flow cytometry, and compared to parental cells (no NK cell exposure). Bar charts represent the number of CD112/CD155 double positive cells after 5 d in the presence or absence of NK cell exposure. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Selection, Labeling, Flow Cytometry, Titration