Journal: Cell Reports Methods

Article Title: Sequence enrichment profiles enable target-agnostic antibody generation for a broad range of antigens

doi: 10.1016/j.crmeth.2023.100475

Figure Lengend Snippet:

Article Snippet: CD130 , Sino Biological , Cat# 10974-HCCH.

Techniques: Produced, Recombinant, Sequencing, Software

Journal: Biomacromolecules

Article Title: Development of CD33-Targeted Dual Drug-Loaded Nanoparticles for the Treatment of Pediatric Acute Myeloid Leukemia

doi: 10.1021/acs.biomac.4c00672

Figure Lengend Snippet: Preparation and characterization of CD33 NPs. (A) Schematic overview of the antibody–nanoparticle conjugation process, using maleimide–thiol chemistry. (B) Table summarizing conjugated NPs characteristics in terms of drug loading, amount of antibody conjugated obtained via Micro BCA, DLS-measured hydrodynamic diameter (nm), and polydispersity index (PdI) values, and PALS-measured zeta potential values. Data are presented as mean ± SD, from measurements performed in triplicate and averaged from at least n = 3. (C) TEM images of CD33 NPs, representative of two independent experiments.

Article Snippet: FLISA studies were performed as previously described, using recombinant human CD33-Fc (Sino Biological) at 1 μg/mL to coat the plate wells.

Techniques: Conjugation Assay, Zeta Potential Analyzer

Journal: Biomacromolecules

Article Title: Development of CD33-Targeted Dual Drug-Loaded Nanoparticles for the Treatment of Pediatric Acute Myeloid Leukemia

doi: 10.1021/acs.biomac.4c00672

Figure Lengend Snippet: Binding of nanoformulations to recombinant CD33-Fc and CD33-expressing cells. (A–D) Binding of rhodamine 6G-loaded NPs to recombinant CD33-Fc in FLISA assays: (A) dose-dependent binding, (B) binding of NPs (50 μg polymer/mL) ± preincubation with CD33-Fc (10 μg/mL), (C) binding of NPs (500 μg polymer/mL) ± preblock with CD33 mAb (40 μg/mL), (D) binding of NPs (500 μg polymer/mL) in competition with varying concentrations of gemtuzumab (0.00256–40 μg/mL). Data are presented as mean ± SD, n = 3. (E) Binding of nonfluorescent nanoformulations to CD33-Fc and IgG-Fc evaluated by SPR: (i) binding of the NPs at 4 mg/mL to CD33-Fc and IgG-Fc, (ii) binding of CD33 NPs to CD33-Fc at varying concentrations, with linear regression and corresponding goodness of fit ( R 2 ). Binding is presented as response relative to baseline observed 5 s before the end of the injection period. Data are presented as mean ± SD, n = 2. (F) Cells were treated with blank CD33 or nude NPs (750 μg polymer/mL) for 1 h at 4 °C. Then, cells were washed, stained with PE-labeled anti-CD33 antibody or isotype control antibody, and PE-fluorescence was analyzed by flow cytometry. Representative histograms are shown for each condition tested, (i), as well as the corresponding reduction of fluorescence compared with the positive stained control observed after treatment with the NPs for each cell line (ii). (G) Confocal microscopy images of MOLM-13 cells treated with 500 μg polymer/mL rhodamine 6G-loaded NPs for 1 h at 4 °C, followed by a washing step and a further 2 h-incubation at 37 °C. Scale bar is 50 μm, and blue and red staining denote cell nuclei and nanoparticles, respectively. Representative data from n = 2.

Article Snippet: FLISA studies were performed as previously described, using recombinant human CD33-Fc (Sino Biological) at 1 μg/mL to coat the plate wells.

Techniques: Binding Assay, Recombinant, Expressing, Fluorophore-linked Immunoabsorbent Assay, Polymer, Injection, Staining, Labeling, Control, Fluorescence, Flow Cytometry, Confocal Microscopy, Incubation

Journal: Biomacromolecules

Article Title: Development of CD33-Targeted Dual Drug-Loaded Nanoparticles for the Treatment of Pediatric Acute Myeloid Leukemia

doi: 10.1021/acs.biomac.4c00672

Figure Lengend Snippet: Targeted delivery of synergistic drug combination within dual CD33 NPs. (A) MV4-11, (B) MOLM-13, and (C) MOLM-14 cells were treated with blank or dual-loaded conjugated (CD33 NP) or nonconjugated (nude NP) nanoparticles for 1 h at 4 °C. Then, cells were washed, counted, and reseeded for 72 h of incubation at 37 °C, prior to measurement of cell viability. Where appropriate, cells were preincubated with 5 μg of free-gemtuzumab for 15 min at 4 °C and washed, prior to NP incubation. Data are presented as mean ± SD, n = 3.

Article Snippet: FLISA studies were performed as previously described, using recombinant human CD33-Fc (Sino Biological) at 1 μg/mL to coat the plate wells.

Techniques: Incubation

Journal: Cellular immunology

Article Title: Functional analysis of acquired CD28 mutations identified in cutaneous T cell lymphoma

doi: 10.1016/j.cellimm.2017.07.002

Figure Lengend Snippet: A–C) Purified CD28-deficient CD4+ T cells were retrovirally transduced with either empty vector (RV), CD28-F51I, CD28-F51V or the CTLA4-CD28 chimera and cocultured with CHO cells expressing either IAd alone (panel A), or coexpressing human CD80 (panel B) or human CD86 (panel C) and stimulated with OVA peptide at the indicated doses. D and E) Purified CD4+ cells as described above were stimulated with plate bound anti-CD3 (.01 μg/ml) alone or in combination with graded doses of plate bound human CD80Ig or CD86Ig and proliferation determined. For all panels, the data shown are the mean ± standard deviation of triplicate wells and are representative of 3–5 independent experiments. Statistical comparisons were made comparing the mutant constructs to the wild type construct, and the wild type to empty vector. **= p<.001 by 2 tailed, multiple comparison, unpaired T testing. Differences were significant at all doses.

Article Snippet: Chinese Hamster Ovary cells (CHO) expressing IA d (gift from Dr K. Murphy, Washington University School of Medicine, St Louis, MO) were stably transfected with plasmids expressing either human CD80 or CD86 (Sino Biologicals, China).

Techniques: Purification, Transduction, Plasmid Preparation, Expressing, Standard Deviation, Mutagenesis, Construct

Journal: Cellular immunology

Article Title: Functional analysis of acquired CD28 mutations identified in cutaneous T cell lymphoma

doi: 10.1016/j.cellimm.2017.07.002

Figure Lengend Snippet: A) Retrovirally transduced CD28-deficient splenocytes were stimulated with OVA (0.03 μM) alone or in the presence of CTLA4Ig at doses ranging from 1–1000 μg/ml and proliferation determined. The percent inhibition at each dose of CTLA4Ig was calculated relative to proliferation in the absence of CTLA4Ig. Results shown are representative results from 3 independent experiments. B) Cells were stimulated as above with the addition of either anti-CD80 or anti-CD86 antibody (10 μg/ml), and proliferation measured. *= p <.05 by 2-tailed t-test adjusted for multiple comparisons, comparing anti-CD86 treatment to either OVA alone or OVA plus anti-CD80. No other comparisons were significantly different. Results shown are representative results from 3 independent experiments.

Article Snippet: Chinese Hamster Ovary cells (CHO) expressing IA d (gift from Dr K. Murphy, Washington University School of Medicine, St Louis, MO) were stably transfected with plasmids expressing either human CD80 or CD86 (Sino Biologicals, China).

Techniques: Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Immunochemical engineering of cell surfaces to generate virus resistance

doi: 10.1073/pnas.1702764114

Figure Lengend Snippet: Schematic representation of selection of Rhinovirus blocking, ICAM-binding antibodies. (A) Predicted structure and dimension of ICAM-1 ectodomain and the membrane tethered antibody. (B) The construct used to express antibody on the cell surface. (C) Phage displayed followed by the functional selection to identify the HRV-blocking antibodies. (D) Three models of proposed interactions between ICAM and membrane-tethered antibodies.

Article Snippet: Recombinant human ICAM-1 ectodomain (Sino Biological) was biotinylated with EZ-link Sulfo-NHS-SS-Biotin (Thermo Fisher).

Techniques: Selection, Blocking Assay, Binding Assay, Construct, Functional Assay

Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet: (A) Schematic of possible CD97 signaling mechanisms tested. Listed in red are the methods for testing. (B) Phosphoproteomic data collected from GBM samples identify five phosphorylation sites on the CD97 cytosolic C terminus. The heatmap depicts the percentage of GBM samples with the detected phosphorylation site. (C) Homogenous time-resolved fluorescence (HTRF) ratios from a β-arrestin recruitment assay performed after overexpression of WT CD97 or the ΔPS mutant. Two PDGCs (n = 2 for each) are compiled (n = 4 per condition; unpaired t test; ns p > 0.05; *p < 0.05; **p < 0.01). (D) Immunoblot for p-ERK1/ERK2 after overexpression of WT CD97 or the ΔPS mutant. (E) Bar graphs displaying densitometry ratios from (D) (n = 7; paired t test; ns p > 0.05; *p < 0.05). Two PDGCs (n = 3–4 for each) are compiled. (F) Bar graphs depicting the number of tumorspheres in a tumorsphere formation assay after CD97 knockdown followed by overexpression of shRNA-resistant forms of WT CD97 or the ΔPS mutant. The SCR shRNA groups used for normalization are not shown (n = 4 per PDGC; ANOVA F 5,12 = 80.79, p < 0.0001; Tukey’s multiple comparisons test: EV vs. WT: ***p < 0.001; EV vs. ΔPS: ns p > 0.05; WT vs. ΔPS: *p < 0.05). (G‒I) Single-cell RNA-/ATAC-seq data from showing expression of CD97 , CD55 , and THY1/CD90 . (J) A bar graph depicting the percentage of positive cells measured by flow cytometry after surface staining of PDGCs with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD55, THY1/CD90, and an IgG control (n = 3 for each PDGC; two-way ANOVA F 2,20 = 95.11, p < 0.0001; Tukey’s multiple comparisons test: IgG vs. CD55: **p < 0.01; IgG vs. CD90: ****p < 0.00001; CD55 vs. CD90: ****p < 0.01). (K) Immunoblot for p-ERK1/ERK2 from CD97-overexpressing PDGCs plated on laminin or recombinant forms of putative ligands CD55 and THY1/CD90. (L) Quantification of densitometry ratios from immunoblots in (K) (n = 2–4 per PDGC; EV p-ERK/ERK: two-way ANOVA F 2,6 = 0.9450, p > 0.05; EV ERK/GAPDH: two-way ANOVA F 2,6 = 1.047, p > 0.05; CD97 overexpression p-ERK/ERK: two-way ANOVA F 2,16 = 12.48, p < 0.001; Tukey’s multiple comparisons test: laminin vs. CD55: ns, p < 0.05; laminin vs. CD90: ***p < 0.001; CD97 overexpression ERK/GAPDH: two-way ANOVA F 2,16 = 2.257, ns p > 0.05). Error bars indicate SEM.

Article Snippet: Thy1/CD90 Protein, Human, Recombinant , SinoBiological , 16897-HCCH.

Techniques: Fluorescence, Over Expression, Mutagenesis, Western Blot, Tube Formation Assay, shRNA, Expressing, Flow Cytometry, Staining, Recombinant

Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet:

Article Snippet: Thy1/CD90 Protein, Human, Recombinant , SinoBiological , 16897-HCCH.

Techniques: Recombinant, Modification, Disruption, Activation Assay, Knock-Out, shRNA