Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed in BM-DCs and LECs and supports DC transmigration. ( A ) Flow cytometry analysis of immature (−LPS) and LPS-matured (+LPS) BM-DCs (gated on live/single cells). ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression of 11 independent experiments. ( C – F ) FACS analysis of CD112 expression in ( C ) LPS-matured BM-DCs and ( E ) primary LN-LECs, derived from WT and CD112 KO mice. ( D , F ) Summary of the ∆MFI values of CD112 expression of 4–6 independent experiments. Data points of the same experiment in ( B , D , F ) are connected by a line, and the mean ΔMFI values are indicated by horizontal lines. ( G ) Set up of the transmigration experiments to investigate the transmigration of BM-DCs (WT or KO) across an LEC monolayer (WT or KO). ( H ) Impact of ICAM-1 blockade on transmigration of WT BM-DCs. ( I,J ) Impact of loss of CD112 in either ( I ) LECs or ( J ) BM-DCs on transmigration. ( K ) Impact of simultaneous loss of CD112 in LECs and BM-DCs on transmigration. For each condition in ( H – K ), one representative experiment with n = 3 technical replicates is shown on the left, and a summary of the averages of 4 independent experiments (biological replicates, each experiment in a different color) is shown on the right. Data points of the same experiment are connected by a line. ( L ) Adhesion assay of WT and KO BM-DCs to WT or KO lymphatic endothelium. The pool of two independent experiments with three replicates per condition is shown (each dot represents a sample). # BM-DCs: number of BM-DCs. Data in all graphs show mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Transmigration Assay, Flow Cytometry, Staining, Expressing, Derivative Assay, Cell Adhesion Assay

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 expression is high in LECs but low in DCs present in murine skin. ( A , B ) FACS analysis was performed to detect CD112 expression in dermal LECs and BECs. ( A ) Depiction of the gating strategy in one representative experiment. ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression observed in 5 independent experiments. ( C – G ) Impact of TPA-induced skin inflammation on the expression of CD112 in LECs. ( C ) Schematic depiction of the experiment: Inflammation was induced in the murine ear skin by topical application of TPA and the ear skin and draining auricular LNs analyzed 24 h later. ( D – G ) FACS analyses were performed to quantify CD112 expression levels in LECs present in control or inflamed tissues. ( D , E ) Analysis of murine ear skin and ( F , G ) auricular LN single-cell suspensions. ( E , G ) The summary of ∆ MFI values was recorded in 5–6 different experiments performed in one control (CTL) and one TPA-inflamed (TPA) ear skin. ( H , I ) FACS gating and quantification of CD112 expression in DCs present in CTL and TPA-inflamed ear skin. ( H ) Gating strategy and ( I ) summary of ∆MFI values recorded in 3 different experiments. ( J – P ) Crawl-out experiments. ( J ) Schematic depiction of the experiment performed to evaluate CD112 expression in ( K – M ) DCs that had emigrated from murine ear skin into the culture medium or in ( N – P ) DCs that had remained in the cultured ear skin at the end of the experiment. Representative ( K , N ) FACS dot plots (gating on single/live cells), identifying DCs as MHCII + CD11c + cells. ( L , O ) Representative histogram plots showing CD112 expression in WT and KO DCs as well as the corresponding fluorescence minus one (FMO) control. ( M , P ) Summary of ∆MFI values (defined as specific staining—FMO) recorded in 4 different experiments performed with one WT and one KO mouse each. Data points in ( B , E , G , I , M , P ) of the same experiment are connected by a line.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Staining, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Loss of CD112 does not impact the in vivo migration of adoptively transferred or endogenous DCs to dLNs. ( A – D ) Adoptive transfer experiment. ( A ) Scheme of the experiment. ( B ) Gating strategy to identify fluorescently labeled adoptively transferred BM-DCs in popliteal LNs. ( C ) The ratio of KO–WT DCs recovered from popliteal LNs draining control (CTL) or CHS-inflamed (CHS) footpads of WT or KO mice. ( D – J ) FITC painting experiment. ( D ) Scheme of the experiment. ( E ) ΔEar thickness, defined as the difference between the ear thickness measured at the start and at the end of the experiment. ( F ) Cellularity and ( G ) weight of the ear-draining auricular LN at the end of the experiment. ( H ) Gating strategy to identify and quantify the number (#) of ( I ) all CD11c + MHCII hi migratory DCs (mDCs) and ( J ) FITC + mDCs. Summaries of three ( A – D ) and two ( D – J ) independent experiments, each with 2–7 mice per condition, are shown. Each dot represents one mouse. Mann–Whitney t -test was used. Red bars in all graphs show the mean. ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vivo, Migration, Adoptive Transfer Assay, Labeling, Control, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Derivative Assay, Blocking Assay, Control, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Gut-primed neutrophils activate Kupffer cells to promote hepatic injury in mouse sepsis

doi: 10.1038/s41467-025-65572-8

Figure Lengend Snippet: WT mice were subjected to CLP and the small intestines were harvested after 20 h. A The number (no.) of neutrophils in the gut epithelium. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 4 samples/group) and compared by paired two-tailed Student’s t test. P value: 1.11E-2. *p < 0.05 vs. Sham. B Representative microscopic images of the gut epithelium (original magnification, ×630; scale bar: 10 μm). Experiments were performed 3 times, and representative images are shown. Bone marrow - derived neutrophils (PMNs) were treated with or without LPS in the presence and absence of intraepithelial lymphocytes (IELs) at 1:1 ratio for 12 h to evaluate NETs. C Representative microscopic images from 3 independent experiments are shown (original magnification, ×200; scale bar: 100 μm). Ly6G (purple) serves as a neutrophil marker. D Representative dot plots following the gating strategy of NETs in Fig. and E frequency of NETs + (Cit-H3 + MPO + ) neutrophils assessed by flow cytometry are shown. Experiments were performed 3 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 6 samples/group) and compared by one-way ANOVA and SNK test. P values based on the order of appearance: 3.02E-7, 1.42E-6, 4.15E-5. *p < 0.05 vs. PBS, # p < 0.05 vs. LPS, † p < 0 . 05 vs. PBS+IELs. F The number of CD112 + neutrophils in the gut epithelium of sham and CLP mice. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 4 samples/group) and compared by paired two-tailed Student’s t test. P value: 1.11E-2. *p < 0.05 vs. Sham. Neutrophils were incubated with IELs and LPS in the presence of a vehicle (PBS, Veh.), isotype control (Iso.), or 10 μg/mL anti-CD112 Ab for 12 h to evaluate NETs by flow cytometry. G Representative dot plots following the gating strategy of NETs in Fig. and H frequency of NETs + neutrophils are shown. Experiments were performed 2 times, and all data were used for analysis. Data are expressed as mean ± SEM (n = 5 samples/group) and compared by one-way ANOVA and SNK test. P values based on the order of appearance: 6.21E-3, 2.52E-2. *p < 0.05 vs. Veh., # p < 0.05 vs. Iso. CLP cecal ligation and puncture.

Article Snippet: Neutrophils incubated with IELs and LPS were also treated with a vehicle (PBS), isotype control, or 10 μg/mL anti-CD112 Ab (Cat. No.: MAB3869; R&D Systems, Minneapolis, MN).

Techniques: Two Tailed Test, Derivative Assay, Marker, Flow Cytometry, Incubation, Control, Ligation

Journal: Nature Communications

Article Title: Gut-primed neutrophils activate Kupffer cells to promote hepatic injury in mouse sepsis

doi: 10.1038/s41467-025-65572-8

Figure Lengend Snippet: During sepsis, neutrophils interact with IELs via CD112 in the gut, resulting in increased NETosis via PAD4-mediated histone citrullination (Cit). NET-forming neutrophils migrate from the gut into the liver and activate PAR-1 on Kupffer cells by NET-contained proteases, such as NE. NET-activated Kupffer cells polarize to an iNOS-expressing M1 phenotype and produce proinflammatory mediators, such as IL-6 and TNF. This gut-liver crosstalk mediated by immune cells leads to sepsis-induced liver injury. PAD4, protein-arginine deiminase type-4; NET, neutrophil extracellular trap; PAR-1, protease-activated receptor-1; NE, neutrophil elastase; iNOS, inducible nitric oxide synthase. Created in BioRender. Murao, A. (2025) https://BioRender.com/fhkfis3 .

Article Snippet: Neutrophils incubated with IELs and LPS were also treated with a vehicle (PBS), isotype control, or 10 μg/mL anti-CD112 Ab (Cat. No.: MAB3869; R&D Systems, Minneapolis, MN).

Techniques: Expressing

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: 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: 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: 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: 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: 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