anti-hcd45 antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Ramos B cell signaling is inhibited by HA-49K orthologs to comparable levels. Ramos B cell signaling was determined to assess the inhibitory potential of HA-49K orthologs in B cells. Previous incubation of Ramos cells with cell supernatants (red) containing various HA-49K orthologs was performed. Unstimulated cells (unstim.) were used as a negative control (grey), co-incubation with unreactive A549 supernatant (A549) served as a positive control indicated in blue. Cells were washed and stimulated via receptor cross-linking using α-λ Abs. The cellular calcium-response was detected by flow cytometry and the mean peak Ca2+-levels (columns) of 3 individual experiments (dots) including standard deviation are shown. Statistical differences compared to A549 were analyzed using two-way ANOVA test (A) . Immunoblot analysis of pErk1/2 levels upon BCR stimulation of Ramos B cell lines with 1 µg/ml α-λ Abs for 2 min. Unstimulated cells (unstim.) served as negative control while α-λ treated cells (pos. crtl.) and co-incubation with A549 supernatant (A549) served as positive controls. Detection of β-actin levels was used as loading control. One representative blot for Ramos and CD45-/- Ramos cells is shown (B) . The relative detection levels of pErk1/2 to β-actin ratios in Ramos cells were normalized to the positive ctrl. The mean (columns) of 3 individual experiments (dots) including standard deviation is shown. Statistical differences to the positive ctrl. were analyzed using the two-way ANOVA test. Only significant results were indicated in the panel (C) . A two-fold dilution series of cell supernatants containing HA-49K-D64 (D) and purified HA-49K-D64 proteins starting at 8 µg per sample (E) was performed. Samples were either supplemented with 0.5 µg per sample hCD45-Fc (+hCD45-Fc, red) or without (hCD45-Fc, blue). After a 1 h incubation period, the binding of HA-49Ks to Ramos cells was detected using α-HA-based flow cytometry. Undiluted supernatant from untransfected A549 cells (A549) or the 8 µg MT protein were utilized as negative controls and shown as single values at the end of the x-axis separated by the grey dashed line. The mean MFI of 3 individual experiments is displayed for each, including standard deviation in the form of error bars. Erk1/2 phosphorylation was analyzed to investigate the prevention of the inhibitory effect HA 49K-D64 by hCD45-Fc receptors via immunoblotting. The supernatant containing HA-49K-D64 proteins was diluted 1:10 and 4 µg purified HA-49K-D64 proteins were incubated for 1 h with 500 ng hCD45-Fc decoy receptors as indicated in the figure. Subsequently, reagents were incubated with Ramos cells for 1 h Cells were lysed after BCR stimulation with 2 µg/ml α-λ Abs for 2 min. Sample loading was controlled by the detection of β-actin levels. One representative blot is presented (F) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Incubation, Negative Control, Positive Control, Flow Cytometry, Standard Deviation, Western Blot, Control, Purification, Binding Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: HA-49Ks also efficiently suppress the phosphorylation of signaling components during BCR stimulation in primary human B cells. Primary human B cells were isolated from PBMCs (n=4 donors, each depicted as single dots). B cells were treated with HA-49K orthologs (red) or left untreated (used as positive control, blue) and stimulated with 10 µg/ml α-IgG Abs. Unstimulated cells and 1 µg MT treated cells, as an unrelated protein control, functioned as negative controls (grey). The number of cells positive for phosphorylated Syk was measured by flow cytometry. To identify CD19+ live cells in PBMCs, cell fraction was selected and debris excluded using the forward scatter area (FSC-A) vs the sideward scatter area (SSC-A). Cell doublets that deviated from the linear correlation between the FSC-A and the forwscatter height (FSC-H) were excluded from downstream analysis. Identification of live cells was done by gating for FSC-A vs live/dead staining. B cells were identified using FSC-A vs CD19 plots. The columns indicate mean MFIs measured for B cells using the anti-phosphorylated Syk staining with error-bars depicting the standard deviation. Statistical analysis was performed using the two-way ANOVA test. Only significant differences (****P<0.0001) to the positive control are indicated in the panel (A) . From this setting lysates of primary B cells were generated and examined by immunoblotting for the phosphorylation of Syk (B) and Erk1/2 (C) . Total Syk and IgG served as a loading controls. One representative blot of each is shown.

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Isolation, Positive Control, Control, Flow Cytometry, Staining, Standard Deviation, Generated, Western Blot

Journal: Frontiers in Immunology

Article Title: Inhibition of B cell receptor signaling induced by the human adenovirus species D E3/49K protein

doi: 10.3389/fimmu.2024.1432226

Figure Lengend Snippet: Only HAdV-D infected A549 cells bind soluble hCD45-Fc. A549 cells were infected with HAdV-A12, -B7, -B35, -C5, -D8, -D19, -D36, -D64 and -D64ΔE3 and -E4, viruses with an MOI of 5 for 24 h Efficient infection was confirmed by internal hexon protein expression (red) using 2Hx-2 mAbs in comparison to isotype control staining (grey) of infected A549 cells in flow cytometry. Displayed are representative histograms of productive infections (A) . Infected cells were treated with +/- (red/blue) 0.5 µg hCD45-Fc per sample. Bound CD45 molecules were detected by CD45-ECD staining using α-human pan-CD45 MEM-28 in flow cytometry. Mock infected cells as well as cells infected with HAdV-D64ΔE3 virus served as negative controls. As positive control the cell clone stably expressing HA-49K of HAdV-D64 was applied. The mean of 3 individual experiments (columns) from (dots) including standard deviation, presented as error bars, is shown. Significant differences between +/- hCD45-Fc treatment were determined using the two-way ANOVA test and are indicated in the panel (B) .

Article Snippet: To quantify the binding activity to human CD45, infected cells or HA-49K producer cell lines were treated with 0.5 µg/sample of recombinant human CD45-ECD with an IgG1 Fc-tag (hCD45-Fc) (Sino Biological).

Techniques: Infection, Expressing, Comparison, Control, Staining, Flow Cytometry, Virus, Positive Control, Stable Transfection, Standard Deviation

Journal: bioRxiv

Article Title: TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

doi: 10.1101/2024.03.05.583596

Figure Lengend Snippet: Diagram depicting timelines for in vivo experiments. b. Engraftment in blood, bone marrow and spleen in NSG cohorts, measured as the proportion of human cells (hCD45+). c. Engraftment in blood, bone marrow and spleen in SGM3 cohorts, measured as the proportion of human cells (hCD45+). d. Percentage of myeloid, B cell lymphoid, T cell lymphoid and HSPC in the bone marrow of NSG cohorts. e. Percentage of myeloid, B cell lymphoid, T cell lymphoid and HSPC in the bone marrow of SGM3 cohorts. N/A: Not analyzed. Empty circles = SGM3. Filled circles = NSG. Data are represented as mean ± standard deviation. Statistical comparisons were performed using one way ANOVA followed by a Tukey’s Honestly-Significant Difference post-hoc test between each two groups; Tukey’s p values are only shown if ANOVA was significant; ns= non-significant, *p≤0.05, **p≤0.01.

Article Snippet: For neurons and neuroglia from both eGFP-edited and IDUA- edited mouse cohorts, the following antibody panel was used: hCD45-PE-Cy7 (Clone: REA747; Miltenyi), mCD45-APC-Cy7 (Clone: 30-F11; BD Biosciences), and CD11b-PE (Clone: M1/70; Biolegend).

Techniques: In Vivo, Standard Deviation

Journal: bioRxiv

Article Title: TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

doi: 10.1101/2024.03.05.583596

Figure Lengend Snippet: Edited HSPCs as a vehicle for therapeutic delivery across the blood-brain barrier. a. Engraftment in brain, measured as the proportion of human cells (hCD45+) in the leukocyte compartment in NSG and SGM3 animals injected with HSPC edited with the eGFP or IDUA cassettes. b. Percentage of CD11b + among the human cells from animals injected with HSPC edited with the eGFP or IDUA cassettes. c. Percentage of eGFP+ cells within the human fraction of cell isolated from the brain of animals engrafted with HSPC edited with eGFP cassettes. d. Percentage of eGFP+ cells within the human CD11b + and CD11b - fractions from the brain of animals engrafted with HSPC edited with eGFP cassettes. e. Percentage of edited alleles among human cells found in the brain of animals engrafted with HSPC edited with IDUA cassettes. Empty circles = SGM3. Filled circles = NSG. Data are represented as mean ± standard deviation. Statistical comparisons were performed using unpaired T tests when comparing two groups , or one way ANOVA when comparing three groups followed by a Tukey’s Honestly- Significant Difference post-hoc test between each two groups ( , 5b). Tukey’s p values are only shown if ANOVA was significant; ns= non-significant, *p≤0.05.

Article Snippet: For neurons and neuroglia from both eGFP-edited and IDUA- edited mouse cohorts, the following antibody panel was used: hCD45-PE-Cy7 (Clone: REA747; Miltenyi), mCD45-APC-Cy7 (Clone: 30-F11; BD Biosciences), and CD11b-PE (Clone: M1/70; Biolegend).

Techniques: Injection, Isolation, Standard Deviation