Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from . P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from ). P-values were determined by two-sided Student’s t-test.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Infection, Quantitative Proteomics, Control, Virus, Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 aging” (A) and “CD47 hypertension” (B). C) Overview figure of the data derived from the literature searches. Age-related increased CD47 levels may contribute to pathogenic conditions associated with severe COVID-19.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay, Infection

Journal: Blood

Article Title: Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages.

doi: 10.1182/blood-2009-07-231449

Figure Lengend Snippet: Figure 1. Galectin-5 is present on the surface of rat red cells. (A) Freshly isolated reticulocytes or erythrocytes were adsorbed on glass coverslips and processed for immunofluorescence as described in “Fluorescence-activated cell-sorting analysis of exosomes and red cells, fluorescence microscopy of red cells.” Transmission images of red cells (left) and corresponding fluorescence imaging (right) were recorded on cells by the use of purified rabbit anti–galectin-5 antibody followed by incubation withAlexa 488 anti–rabbit antibody. (B) Young reticulocytes and erythrocytes were analyzed by flow cytometry by the use of antibodies raised against Gal-2 (dashed line), Gal-4 (dotted line), and Gal-5 (solid line), already tested for their specificity (top), or the produced anti–galectin-5 serum (solid line) and the preimmune serum (bottom, dashed line). Tinted patterns indicate cell labeling obtained in the absence of primary antibodies. (C) Lymphocytes isolated from rat blood, as described in “Cells,” were analyzed by flow cytometry for Gal-5 (left, solid line), CD47 (middle, solid line) and Syto 16 green (right, solid line). Tinted patterns indicate cell labeling in the absence of primary antibodies. (D) Ghost and raft extracts isolated from reticulocytes or mature erythrocytes, as described in “Red cell subcellular fractionation,” were processed by SDS-PAGE and analyzed by Western blot for the indicated proteins. The molecular mass (kDa) standards are indicated on the left.

Article Snippet: Mouse anti–rat CD47 was from Serotec Limited.

Techniques: Isolation, Fluorescence, FACS, Microscopy, Transmission Assay, Imaging, Incubation, Cytometry, Produced, Labeling, Fractionation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Histograms depicting cell surface expression of CD24 and CD47 by flow cytometry on mouse cancer cell lines. B, Correlation of CD24 and CD47 surface expression of cell lines shown in A by geometric MFI. Data shown as mean ± SD of 3 technical replicates. Simple linear regression was performed to assess correlation. C, Representative plots showing quantification of CD45+ phagocytic primary mouse macrophages co-cultured with CFSE+ KPCA.C. Co-cultures were exposed to vehicle control (PBS) or 10 ug/ml of monoclonal antibodies against mouse CD47, CD24, or the combination for 2 hours. Phagocytosis is represented as CD45+ macrophages that had engulfed CFSE+ KPCA.C cells as a percentage of the total macrophage population. D, Quantification of phagocytosis for cell lines in A . Cell lines are organized based on expression levels of each surface marker. Data represent mean ± SD of 3 technical replicates. E, Correlation of cell surface expression levels of CD47 and CD24 compared to phagocytosis upon treatment with the corresponding antibodies for each cell line. Data points depict mean ± SD from 3 replicates for each experiment. Correlation was assessed by simple linear regression. F, Representative microscopy images of GFP+ KPCA.C cells when co-cultured with primary mouse macrophages upon treatment with vehicle control (PBS), 10 ug/mL anti-CD47, 10 ug/mL anti-CD24, or the combination for 6.5 days. Top row depicts raw images of GFP+ fluorescence. Bottom row depicts purple GFP+ mask for above images used for quantification of cancer cell growth. Scale bar, 800 µm. G, Quantification of fluorescent well area from co-culture experiments for multiple cell lines after 6.5 days, organized by surface expression of CD24. Cancer cells were quantified by either green (KPCA.C, 3LL ΔNRAS, MC38) or red (238N1) fluorescent area based on their fluorophore expression. Data and means shown from one (3LL ΔNRAS, MC38) or two (238N1, KPCA.C) independent experiments with 3 technical replicates per experiment. D,G, statistical significance ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 determined by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Bioprocessing, Marker, Microscopy, Fluorescence, Co-Culture Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Representative histograms demonstrating cell surface expression of CD47 and CD24 on knockouts of KPCA.C and knockdowns of 238N1 by flow cytometry. B, Representative gating of phagocytic APC CD45+ mouse macrophages when co-cultured with the indicated CFSE+ KPCA.C knockouts treated with vehicle control (PBS) for 2 hours. Phagocytic macrophages are calculated as CD45+ cells that have engulfed CFSE+ cancer cells after 2 hours as a percent of all macrophages. C,D, Quantification of phagocytosis as a percentage of the maximum phagocytic response of macrophages using KPCA.C knockout cells ( C ) or 238N1 knockdown cells ( D ) treated with vehicle control (PBS), anti-mouse CD47 antibody, anti-mouse CD24 antibody, or the combination. Data represents mean ± SD of 3 technical replicates. E,F, Quantification of fluorescent well area as a measure of GFP+ KPCA.C knockout cells ( E ) or mCherry+ 238N1 knockdown cells ( F ) growth after co-culture with primary mouse macrophages and the indicated antibodies on day 6.5. Data represents mean ± SD from two independent experiments of 3 technical replicates each. G,H, Quantification of phagocytosis using CFSE+ MC38 ( G ) or 3LL ΔNRAS ( H ) cancer cells that overexpress CD24 after co-culture with primary mouse macrophages and the indicated antibodies. Data represent mean ± SD from 3 individual experiments each containing 3 technical replicates. I, Quantification of phagocytosis using StayGold+ KPCA.C cancer cells treated with vehicle control (PBS), or anti-mouse CD24 antibody, in the absence or presence of FcR blocking reagents (Fc1, anti-mouse Truestain clone 93; Fc2, anti-mouse CD16/CD32 clone 2.4G2). Data represents mean ± SD of 3 technical replicates. ( C-H ) ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Knock-Out, Knockdown, Co-Culture Assay, Blocking Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: Results of scRNA-seq of sorted CD45+ immune cells from experiments using CD24 or CD47 knockout tumors. ( A,C,E ) Comparison of CD47- tumors (KPCA.C CD47 knockout, 238N1 CD47 knockout) to wild-type tumors (KPCA.C control, 238N1 control). A, Relative frequencies of immune cells from CD47- versus wild-type tumors. C, UMAP showing identified cell clusters. E, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways. ( B,D,F ) Comparison of CD24- tumors (KPCA.C CD24 knockout, 238N1 CD24 knockout) to wild-type tumors (KPCA.C control, 238N1 control). B, Relative frequencies of immune cells from CD24- versus wild-type tumors. D, UMAP showing identified cell clusters. F, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Knock-Out, Comparison, Control

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Diagram showing process for high-throughput development and functional evaluation of bispecific antibodies targeting macrophage immune checkpoints. Antibody sequences were transformed into scFvs and cloned into a knob-into-hole format using a human IgG1 Fc. Constructs targeting macrophage immune checkpoints (CD47, CD24, SIRPa, PD-1) were cloned into knob formats and crossed with tumor-binding constructs in a hole format. Bispecific antibodies (n = 77) were expressed in Expi293F cells and used for downstream biochemical and functional analysis. B, Growth of StayGold+ DLD-1 cells in co-culture with human macrophages and each bispecific antibody. Each curve represents the mean for an individual bispecific antibody from 4 replicates. Black curve with hashed lines represents mean and 95% CI of control wells . C, Anti-tumor efficacy of bispecific antibodies at approximately t = 6.5 days as evaluated by macrophage checkpoint category. *p<0.05, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. D-F, Growth curves for each of the WTa2d1 constructs ( D ), CD24-3 constructs ( E ), or CV1 constructs ( F ). G, Representative whole-well imaging of co-cultures treated with different bispecific antibodies at approximately t = 6.5 day. Green signal depicts growth of StayGold+ DLD-1 cells. Rows contain different macrophage checkpoint arms, while columns contain different tumor-binding arms. H, Scatter plot showing binding of each bispecific antibody to human neutrophils versus red blood cells. I, Representative histograms showing binding of the indicated bispecific antibodies to human neutrophils and red blood cells.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: High Throughput Screening Assay, Functional Assay, Transformation Assay, Clone Assay, Construct, Binding Assay, Co-Culture Assay, Control, Imaging

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Surface CD47 (A) and ecto-CRT protein expression (B) shown as geometric MFI in a panel of six different NSCLC cell lines. Each histogram represents the mean (± SD) of three to five independent experiments. Comparisons made by ANOVA with Fisher's post hoc multiple comparison analysis. ### p < 0.03, ## p < 0.01, # p < 0.0005. Below each histogram, a matrix table where all p values resulting from post hoc analysis are reported. Expression levels of CD47 (C) and CRT mRNA (D) in 226 untreated primary NSCL adenocarcinomas (GEO accession number GSE31210 ). Middle lines in box plots represent the medians and whiskers represent 5–95% CI ( ### p < 0.03, Kruskal-Wallis test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Modulation by gefitinib of surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Flow cytometric profiles of surface CD47 (A) and ecto-CRT expression (B) on DMSO-treated (CTRL, gray lines) and gefitinib-treated (GEF, red lines) NSCLC cells. Histograms show the mean (± SD) of fold changes of CD47 (C) and ecto-CRT (D) geometric MFI, relative to DMSO-treated controls ( N = 3–5, * p < 0.05, ** p < 0.01 paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Gefitinib-induced CD47 down-regulation promotes tumor cell phagocytosis by dendritic cells. Representative flow cytometric analyses and mean ± SD ( N = 4 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells (see Methods) against PC9 (A,B) , HCC827 (C,D) , and H1975 cells (E,F) treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Cancer cells exposed to the drug for 48 h were labeled with DiO tracer and then co-cultured with dendritic cells for 2 h at a 1:1 ratio. Phagocytosis assays were also run at 4°C as controls. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (* p < 0.05, n.s., not significant, paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Activity Assay, Derivative Assay, Labeling, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Blocking of CD47 on tumor cells induces phagocytosis by dendritic cells. Dendritic cells were co-cultured with DiO tracer-labeled HCC827 (A) and H1975 (B) cancer cells in the presence of IgG isotype control or anti-CD47 mAb as indicated. Shown is the mean (± SD, N = 3 independent healthy donors) percentage increase of CD11c/DiO tracer double positive cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( # p < 0.05, ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Blocking Assay, Cell Culture, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Expression levels of surface CD47 increase in cancer cells acquiring resistance to gefitinib and inhibit tumor cell phagocytosis by dendritic cells. Surface CD47 (A) and ecto-CRT expression (B) in gefitinib-sensitive PC9 and HCC827 (gray lines) and resistant PC9GR and HCC827GR (green lines) cell lines. Representative flow cytometric histograms (left) and mean (± SD, N = 3–5) fold changes of treatment-resistant over sensitive cells (right). (C) Representative flow cytometric histogram plots (left) and mean (± SD) fold changes (right) of surface CD47 levels in resistant cell lines treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Acquisition of resistance to gefitinib abolished drug-induced CD47 down-regulation in PC9GR (* p < 0.05, ** p < 0.01, n.s., not significant, paired two-tailed Student's t -test). (D) Mean ± SD ( N = 3 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells against PC9GR cells in the absence or presence of gefitinib treatment, performed at 4°C as control and at 37°C. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (paired two-tailed Student's t -test. n.s., not significant). (E) Dendritic cells were co-cultured with gefitinib-treated, DiO tracer-labeled PC9GR cells in the presence of IgG isotype control or anti-CD47 mAb. Shown is the mean ± SD ( N = 3 independent healthy donors) percent change of CD11c + /DiO + tracer double positive dendritic cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Two Tailed Test, Activity Assay, Derivative Assay, Control, Cell Culture, Labeling

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: CD47 is overexpressed on malignant lymphocytes in mycosis fungoides (MF) tumors. a A representative image of a skin involved by MF demonstrates intense CD47 staining on atypical TOX + malignant cells. HE, hematoxylin and eosin (20x). b Targeted single-cell RNA transcriptomics as tSNE plots of concatenated tumors from three patients with MF tumors (n = 287 cells total). Clusters called by recursive dendrogram split and annotated from preferentially expressed genes. Tcm, T cell central memory; Tem, T cell effecor memory; DC, dendritic cells. c The intensity of CD47 expression (anti-CD47 antibody-oligo conjugate; AbSeq) over the various cell population defined in Fig. 1b. d Statistical analysis of expression of CD47 (molecules per cells) in different cell populations. *, p < 0.05; ***, p < 0.001 e CD47 expression on CD3 + TOX + MBL2 cells. Flow cytometry of a cell suspension from a primary cell culture. Grey tinted area, an isotype control. Red tinted area, anti-CD47 antibody. f Tumor growth curves of CD47hi WT MBL2 (WT) and CD47 KO MBL2 (CD47 KO) after implantation in B6.SJL mice. n = 5 mice in each group. g Representative imaging of mice 10 days after implantation of CD47hi WT MBL2 (WT) or CD47 KO MBL2 (KO) cells demonstrating large ulcerated tumor in WT, while KO mouse exhibited medium-size tumor without ulceration

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Staining, Expressing, Flow Cytometry, Suspension, Cell Culture, Control, Imaging

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: Anti-CD47 therapy is efficient in controlling the malignant lymphoma growth in a murine model of mycosis fungoides (MF). a Design of anti-CD47 experiments (I.P., intraperitoneal). b Tumor thickness after MBL2 implantation during treatment with anti-CD47 antibody or irrelevant IgG control (n = 5 mice per group). c Percentage of TOX + tumor cells in the inflammatory infiltrate of auricular skin at day 24 after implantation. The percentage of caspase-3 + cells is indicated in black (n = 5 mice per group) (Tx, treatment). d Percentage of F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). e Percentage of MHC class II + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). f Percentage of TNF-α + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). g Percentage of NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). h Percentage of IFN-γ + cells among NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). i Percentage of CD62L + NKG2A-cells among NK1.1. + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). j Schematic of experimental design. k Representative images of mice with tumors after treatment with IFN-α, anti-CD47, or combination anti-CD47 + IFN-α. l The percentage of malignant cells (TOX +) in the TME on day 24 after implantation of MBL2 (n = 5 mice per group). m Representative flow cytometry of CD107a and IFN-γ NK cells (gated on NK1.1 + cells) during therapy with anti-CD47 antibody. n Quantification of CD107a + IFN-γ + NK cells and CD107a + IFN-γ-NK cells (n = 5 mice per group). o The cytotoxic assay of NK cells derived from splenocytes of treated mice co-cultured with MBL2 cells at the indicated effector to target cell ratios

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Control, Flow Cytometry, Derivative Assay, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The effect of anti-CD47 therapy is mediated by cytotoxic NK cells and does not depend on IFN-γ. a Schematic of NK1.1 depletion experiment. b A representative flow showing the percentage of NK cells in non-depleted and NK-depleted mice prior to therapy. c Representative images of mice with tumors 14 days after MBL2 implantation with and without NK1.1 depletion prior to therapy initiation. D The percentage of malignant cells (CD3 + TOX + cells) per ear in non-depleted and NK-depleted mice 24 days after MBL2 implantation. N = 5 mice per group. ***, p < 0.001 e Tumor thickness 24 days after MBL2 implantation in mice treated with irrelevant IgG or anti-CD47 antibody (n = 5 mice per group). f Schematic of experimental design for IFN-γ KO mice. g The volume of lymph nodes 24 days after MBL2 implantation in mice treated with anti-CD47 antibody (n = 3 mice per group). *,p < 0.05; **,p < 0.01; ****,p < 0.0001; ns, non-significant

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The anti-CD47 therapy is accompanied by an influx of NK cells in the TME in patients with relapsed/refractory mycosis fungoides (MF). a Representative images of high and low CD56 number in the dermal infiltrate of tumor MF. b Inverse correlation of the number of CD56 cells per 100 dermal lymphocytes and histoscore of CD47 on the epidermotropic malignant lymphocytes (n = 17, r = – 0.48, p < 0.05). c Representative images of a patient treated with six intra-tumoral injections of 10 mg TTI-621 demonstrate a significant reduction of all tumors and plaques after treatment (I, injected tumor; C, control non-injected tumor). d Multispectral fluorescent immunohistochemistry of CD3, CD56, TOX, and DAPI staining of representative tumors before and after treatment with TTI-621 (60X). e Percentage of malignant cells (CD3 + CD4 + TOX +) and NK cells (CD3-CD56 +) in the TME (n = 11 patients; 4 responders and 7 non-responders). f tSNE analysis of cellular composition in patients with cutaneous T cell lymphoma who had responded to therapy with TTI-621 (n = 4 patients), showing eight distinct clusters of NK cells. g Phenotypic characterization of eight distinct clusters of NK cells depending on the percentage of markers. h Grouping of eight NK cell clusters in two groups based on high vs. dim CD56 expression. i Changes in CD56high NK cells vs. CD56dim NK cells after intra-tumoral injection of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4). j Percentage of NK cells before and after six intra-tumoral injections of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4).. *,p < 0.05; **,p < 0.01

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Injection, Control, Immunohistochemistry, Staining, Expressing

Journal: Oncoimmunology

Article Title: Bendamustine with total body irradiation conditioning yields tolerant T-cells while preserving T-cell-dependent graft-versus-leukemia

doi: 10.1080/2162402X.2020.1758011

Figure Lengend Snippet: BEN-TBI does not result in appreciable donor T-cell phenotypic differences post-transplant when compared to CY-TBI. (a–c) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 TCD-BM from naïve C57BL/6 mice with 3 × 10 6 CellTrace Violet-stained tT from naïve BoyJ mice on day 0. Blood and spleen were collected on day +3. (a) % donor T-cells (CD45.1+) was determined by flow cytometry. Using CBCs determined by HemaVet analysis, absolute number of donor T-cells was calculated. (b) After gating on CD45.1+ cells (representing donor T-cells), CellTrace Violet dilution was analyzed using ModFit software to determine proliferation index. Representative CellTrace Violet dilution is shown. (c) Within the CD45.1+ gate, cells were stratified by CellTrace high (non- proliferative) and CellTrace low (proliferative) and CD25 and CD47 expression were analyzed by flow cytometry. Pooled data from 2 experiments with line at mean are shown, n = 6–7 mice/group. * p < .05 , ** p < .01 . (d-g) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 BM with 3 × 10 6 SC from naïve C57BL/6 mice on day 0. Peripheral blood was collected on days +7, +14, +21, +35, and +70 and stained for CD8, CD4, Tbet, GATA3, RORγt, CD134, CD278, PD-1, TIM3, CTLA-4, and CD272. CBCs were determined and used to calculate absolute cell numbers. Average absolute numbers of cells per μL of blood are shown with SEM. Representative flow plots from day +7 with fluorescence minus one (FMO) controls are shown (e). (d) Pooled data from 4 experiments are shown, n = 19 mice/group. (e-g) Pooled data from 2 experiments are shown, n = 10 mice/group. ** p < .01.

Article Snippet: Antibodies used were anti-mouse H2kb PerCP-eFluor710 (clone AF6-88.5.5.3; 46–5958), CD8α PE-Cy7 (53–6.7; 25–0081), CD4 APC (RM4-5; 50-148-54), FoxP3 APC (FJK-16s; 17–5773), CD45.1 APC (A20; 17–0453), GATA3 PE-Cy7 (TWAJ; 25–9966), CD134 PE-Cy7 (OX-86; 25–1341), FoxP3 PE (150D/E4; 12–4774), CD69 PE-Cy5 (H1.2F3; 15–0691), RORγt APC (AFKJS-9; 17–6988), IFN gamma PE (XMG1.2, 12–7311) (Thermo Fisher Scientific), CD45.1 PE-CF594 (A20; 562452), CD3ε PE-CF594 (145–2 C11; 562286), CD44 BB515 (IM7; 565941), CD44 BV510 (IM7; 563114) (BD Biosciences), CD47 PE-Vio770 (REA170; 130-102-383), TIM-3 VioBright FITC (REA602; 130-109-449), TIM-3 PE (REA602; 130-118-563), CD278 VioGreen (REA192; 130-100-739), CD272 PE (REA224; 130-102-689), CD4 APC-Vio770 (GK1.5; 130-102-786), CD4 VioGreen (GK1.5; 130-102-444) (Miltenyi Biotec), Tbet PE-Dazzle594 (4B10; 644828), CTLA-4 PE-Dazzle594 (UC10-4B9; 106318), CD8α Brilliant Violet 421 (53–6.7; 100738), CCR7 PE-Cy5 (4B12; 120114), PD-1 APC (29F.1A12; 135210), CD25 AlexaFluor700 (PC61; 102024), and TNF-α Brilliant Violet 510 (MP6-XT22, 506339) (Biolegend).

Techniques: Staining, Flow Cytometry, Software, Expressing, Fluorescence

Journal: OncoTargets and therapy

Article Title: Integrated RNA Sequencing and Single-Cell Mass Cytometry Reveal a Novel Role of LncRNA HOXA-AS2 in Tumorigenesis and Stemness of Hepatocellular Carcinoma

doi: 10.2147/OTT.S272717

Figure Lengend Snippet: Purified Antibodies About the Stem-Like Cells Centric Panel

Article Snippet: CD47 , 209Bi , CC2C6 , Fluidigm , 3209004B.

Techniques: Purification

Journal: Brain Communications

Article Title: Cortical CD200–CD200R and CD47–SIRPα expression is associated with multiple sclerosis pathology

doi: 10.1093/braincomms/fcae264

Figure Lengend Snippet: qPCR primers

Article Snippet: CD47 , Serotec (MCA911) , BRIC126 , 1:30 000, 1:800 , 4% PFA.

Techniques: Sequencing

Journal: Brain Communications

Article Title: Cortical CD200–CD200R and CD47–SIRPα expression is associated with multiple sclerosis pathology

doi: 10.1093/braincomms/fcae264

Figure Lengend Snippet: IHC antibodies

Article Snippet: CD47 , Serotec (MCA911) , BRIC126 , 1:30 000, 1:800 , 4% PFA.

Techniques:

Journal: Brain Communications

Article Title: Cortical CD200–CD200R and CD47–SIRPα expression is associated with multiple sclerosis pathology

doi: 10.1093/braincomms/fcae264

Figure Lengend Snippet: Gene and protein expression of CD200, CD47, CD200R and SIRPα in NAGM. Gene expression in NAGM of MS compared with GM of controls as measured with qRT-PCR (MS n = 15, CON n = 26) was ( A ) lower for CD200 , ( B ) comparable for CD47 , ( C ) lower for CD200R and ( D ) comparable for SIRPA . ( E ) Immunofluorescent staining of CD200 and MBP shows that CD200 expression was abundant in NAGM and low in NAWM. Scale bar on the overview image is 1 cm and on the zoomed image it is 250 µm. ( F ) CD200 expression co-localized with MBP + myelin as indicated with arrows; scale bar is set at 10 µm. ( G ) CD200 expression co-localized with NFH + axons as indicated with arrows; scale bar is set at 5 µm. ( H ) CD47 expression was, like CD200, abundant in NAGM and lower in NAWM. Scale bar on the overview image is 1 cm and on the zoomed image is 250 µm. ( I ) CD47 strongly co-localized with some MBP + myelin, as indicated with the arrows, and was sometimes localized adjacent to the myelin, on the outer layer, as indicated with the arrowhead. Scale bar is set at 10 µm. ( J ) Occasionally, CD47 co-localized with NFH + axons, scale bar is set at 5 µm. ( K ) CD200R was expressed by Iba1 + microglia; scale bar is set at 10 µm. ( L ) SIRPα was expressed by Iba1 + microglia; scale bar is set at 10 µm. DAB stainings showed ( M ) mainly extracellular deposition and sporadic expression on neurons for CD200, ( N ) mainly extracellular deposition and sporadic expression on neurons for CD47, ( O ) sporadic positive staining of cells for CD200R, and ( P ) light positive neurons and some darker stained round cells for SIRPα. Scale bars are set at 20 µm. In MS NAGM compared with control GM, there was a ( Q ) lower CD200 OD in cortical Layers 1 and 2 (MS n = 19, CON n = 19), ( R ) comparable CD47 OD (MS n = 31, CON n = 28), ( S ) lower number of CD200R + cells/mm 2 (MS n = 24, CON n = 21), and ( T ) comparable number of SIRPα + cells/mm 2 (MS n = 31, CON n = 25). Box plots indicate the median. * P < 0.05, ** P < 0.01, *** P < 0.001. Significance was tested with a quasi-Poisson generalized linear model.

Article Snippet: CD47 , Serotec (MCA911) , BRIC126 , 1:30 000, 1:800 , 4% PFA.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Staining, Control

Journal: Brain Communications

Article Title: Cortical CD200–CD200R and CD47–SIRPα expression is associated with multiple sclerosis pathology

doi: 10.1093/braincomms/fcae264

Figure Lengend Snippet: Distribution of CD200, CD47, CD200R and SIRPα in GM lesions and perilesional GM. (A) CD200 OD was lower in GM lesions ( n = 16) compared with NAGM and perilesional GM ( n = 19). ( B ) CD47 OD was lower in GM lesions ( n = 24) compared with NAGM ( n = 24). ( C ) There was no difference in number of CD200R + cells/mm 2 in GM lesions ( n = 24) or perilesional GM ( n = 18) compared with NAGM ( n = 18). ( D ) In GM lesions ( n = 17) and perilesional GM ( n = 15), there was an increase in the number of SIRPα + cells/mm 2 compared with the NAGM ( n = 31). Box plots indicate the median. * P < 0.05, ** P < 0.01, *** P < 0.001, # P = 0.05. Significance was tested with a quasi-Poisson generalized linear model, correcting for neuronal density and for multiple testing with FDR.

Article Snippet: CD47 , Serotec (MCA911) , BRIC126 , 1:30 000, 1:800 , 4% PFA.

Techniques:

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Preparation and characterization of anti‐CD47‐PCM@NP. A) Hydrodynamic size and zeta potential of CM vesicles, PLGA cores (NP), PCM@NP, and anti‐CD47‐PCM@NP. Data are means ± SD ( n = 3). B) Colocalization of NP/C6 (green) with DiD‐PCM (red), and the colocalization of FITC‐antibody (green) with DiD‐PCM@NP (red), both assessed by confocal laser scanning microscope (CLSM) (scale bar = 5 µm). C) Transmission electron micrographs of (a) NP, (b) CM vesicle, (c) PCM@NP, (d) Anti‐CD47‐PCM@NP, and (e) multiple anti‐CD47‐PCM@NP. All scale bars = 100 nm. D) SDS‐PAGE protein analysis of NP, PCM@NP, CM vesicles, and cancer cell lysate. Samples were tested at equal protein concentrations. CD47 protein and membrane‐specific protein on the cancer cell membrane were efficiently retained on the extracted membrane vesicles and the PCM@NP, detected by western blot. E) Determination of the antibody labeled by PE loaded on the surface of anti‐CD47‐PCM@NP by flow nanoanalyzer. F) The binding affinity of the antibody to the CM vesicles by surface plasmon resonance (SPR).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Zeta Potential Analyzer, Laser-Scanning Microscopy, Transmission Assay, SDS Page, Membrane, Western Blot, Labeling, Binding Assay, SPR Assay

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Anti‐CD47‐PCM@NP effectively distinguished target cells from CD47‐expressing nontarget cells in vitro through indirect active targeting. A) Anti‐CD47‐PCM@NP avoided the blocking of CD47 on RBC and subsequent phagocytosis thus circumvented the on‐target toxicity of free anti‐CD47 towards RBC through the INTACT strategy. Scale bar = 50 µm. B,C) PCM@NP and anti‐CD47‐PCM@NP efficiently escaped the capture by B) macrophages with enhanced and parallel cellular uptake by C) target 4T1 cells, measured by flow cytometry. The antibody selectively dissociated from the carrier PCM@NP at the presence of 4T1 cells with high expression of D) CD47 in contrast to E) CD47 −/− 4T1 cells, shown by colocalization images and distribution map of PE‐antibody (red) and PCM@NP/C6 (green). Scale bar = 20 µm (multi‐cell images), 2 µm (single‐cell images). F) The schematic diagram of the microfluidic device. The tumor cells were cultured in the cavity of the microfluidic chip till adherence, and then exposed to flowing anti‐CD47 or anti‐CD47‐PCM@NP, and fluorescent images were captured at predetermined time points. G) Free anti‐CD47 sufficiently bound to the surface of 4T1 cells with high expression of CD47 (a). The antibody dissociated from PCM@NP at the presence of 4T1 cells (b), in contrast with CD47 −/− 4T1 group (c) (antibody labeled with FITC, green. PCM@NP labeled with DiD, red). Scale bar = 10 µm. Original movies are shown in Movie S1 (Supporting Information) (a), Movie S2 (Supporting Information) (b), and Movie S3 (Supporting Information) (c), respectively. Data are presented as mean ± SD ( n = 3). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Expressing, In Vitro, Blocking Assay, Flow Cytometry, Cell Culture, Labeling

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Evaluation of the biological functions of anti‐CD47‐PCM@NP in vivo. A) In vivo and ex vivo targeting ability of anti‐CD47‐PCM@NP and anti‐CD47 in tumor‐bearing mice models determined by live imaging. B) The semiquantitative analysis of the ratio of fluorescence intensity (tumor/liver) of ex vivo imaging. C) In vivo biodistribution of coumarin 6 (C6)‐labeled formulations in tumor‐bearing mice models at (a) 2 h, (b) 4 h, (c) 8 h, and (d) 12 h after i.v. injection of NP/C6, PCM@NP/C6 and anti‐CD47‐PCM@NP/C6. Data are presented as mean ± SD ( n = 3) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: In Vivo, Ex Vivo, Imaging, Fluorescence, Labeling, Injection

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: Antitumor efficacy of anti‐CD47‐PCM@NP and mechanistic investigation by CyTOF analysis. A) Representative images and phagocytic index of C57BL/6 bone marrow‐derived macrophages (BMDM) phagocytosing tumor cells following treatment with PCM@NP, anti‐CD47, and anti‐CD47‐PCM@NP. Scale bar = 50 µm. B) Timeline of the anti‐tumor efficacy study on tumor‐bearing mice (red arrows indicate intravenous administrations), and average tumor growth curves and picture of tumor tissues after the treatment. C) Individual tumor growth curves in each group. D) viSNE plot of intratumoral cells in tumor tissues after treatment with saline, PCM@NP, anti‐CD47, anti‐CD47‐PCM@NP and all groups merged. E) Heat map of the surface molecule and functional molecule expression of different subsets of immune cells in tumor tissues from all groups merged. F) tSNE visualization of all samples with the expression of CD4 and CD8a respectively. G) Percentage of cells in each cluster after treatment from each group. Data represented as mean ± SD ( n = 6). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents nonsignificance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Derivative Assay, Saline, Functional Assay, Expressing

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: The INTACT strategy efficiently delivers antibodies to tumors with reduced in vivo toxicity. A) Anti‐CD47‐PCM@NP exhibited no significant influence on red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet (PLT). Data represented as mean ± SD ( n = 3). B–D) Anti‐CD47‐PCM@NP relieved the occurrence of fungal infection during antitumor treatment. B) Experimental timeline and treatments in tumor‐bearing mice (arrows indicate intravenous administrations). At day 14, mice were infected with C. albicans via tail vein injection. C) Colony‐forming units (CFU) on day 7 in the kidneys of infected mouse models ( n = 6). D) The survival rates of infected mice with different treatments ( n = 12). Data represented as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: In Vivo, Infection, Injection

Journal: Advanced Science

Article Title: Overcoming the On‐Target Toxicity in Antibody‐Mediated Therapies via an Indirect Active Targeting Strategy

doi: 10.1002/advs.202206912

Figure Lengend Snippet: The INTACT strategy is adaptive to multiple antibody‐based systems. A) Relative tumor volume growth with anti‐CD47‐PCM@NP/PTX treatment ( n = 6). B–G) The INTACT therapy refined the targeting precision of ADC. B) The diagram of ADC construction: Anti‐CD47 was modified with streptavidin and conjugated with DM1 via the crosslinker BMCC‐biotin. C) The conjugation of ADC was confirmed with SDS‐PAGE. D) Experimental timeline for the anti‐tumor efficacy study and hematology assessments of ADC‐PCM@NP (red arrows indicate intravenous administrations). E) Average tumor growth curves, and picture of the tumor tissues after the treatment ( n = 6). F) Individual tumor growth curves in each group ( n = 6). G) Hematology assessments of red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and platelet (PLT) ( n = 3). Data represented as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; NS represents non‐significance).

Article Snippet: The cells were diluted at 1× 10 6 cells per mL in a culture medium and incubated with 1.0 mg mL −1 of a PE anti‐human/mouse/rat CD47 antibody (E‐AB‐F1016D, Elabscience) or anti‐Neu/ErbB2/HER2 Antibody (SC‐7301, Santa Cruz Biotechnology) and CoraLite488‐conjugated Affinipure coat anti‐mouse IgG(H+L) (SA00013‐1, Proteintech) for 30 min at 4 °C.

Techniques: Modification, Conjugation Assay, SDS Page