cd59  (Bio-Rad)

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Gene expression and cytogenetic clusters in MM. (A) Violin plots showing gene expression levels for TNFRSF13B , CD59 , FCGR2B , SLC44A1 , and CD320 in HD, MGUS, SMM, MM, RRMM. Statistical comparisons were conducted using the Kruskal–Wallis test for all pairwise contrasts. (B) Heatmap of the pseudobulk expression profiles showing normalized expression levels for eight putative proxy markers of cytogenetic aberrations across 54 MM patients. (C) Box plots illustrating the differential expression of TNFRSF13B , CD59 , FCGR2B , SLC44A1 , and CD320 across the four cytogenetic patient clusters. Statistical significance was evaluated using two-sided t -tests for all pairwise comparisons p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Gene Expression, Expressing, Quantitative Proteomics

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Kaplan–Meier survival curves showing the prognostic relevance of mRNA expression levels for CD59, SLC44A1, FCGR2B, TNFRSF13B, and CD320 genes. Cell populations with high expression of each gene are shown in blue, while populations with low expression of the same mRNA are shown in red. Reference datasets (GSE, GEO Series) are indicated at the top of each panel. Prognostic significance was assessed using the Cox proportional hazards model (95% confidence interval CI) and log-rank test, with p < 0.05 considered statistically significant.

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Expressing

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Histogram plots showing the cell surface expression of CD59, CD92, CD267, and CD320 molecules on different MM cell lines, including AMO-1, OPM-2, RPMI8226, U-266, and H929 as measured by flow cytometry analysis using fluorochrome-conjugated antibodies in triplicate experiments. Each panel represents the distribution of expression levels for the indicated marker, allowing comparison of protein abundance across the different cell lines compared to unstained control (in blue).

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Expressing, Flow Cytometry, Marker, Comparison, Quantitative Proteomics, Control

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Histogram plots showing the cell surface expression of CD59, CD92, CD267, and CD320 molecules on malignant plasma cells (PCs) from eight bone marrow samples of newly diagnosed, untreated MM patients as measured by flow cytometry analysis using fluorochrome-conjugated antibodies in triplicate experiments. Each panel represents the distribution of expression levels for the indicated marker across the eight MM patients samples, allowing comparison of protein abundance compared to unstained control (in blue).

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Expressing, Clinical Proteomics, Flow Cytometry, Marker, Comparison, Quantitative Proteomics, Control

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Oligonucleotides used for qPCR analysis.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Sequencing, Amplification

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Antibodies and dyes used.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Concentration Assay

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Graphic representation of relative gene expression of complement factors (CD55, CD59, CFH and CFI) and cytokines (IL-6 and TNFα) in synovial fibroblast cell line K4IM after PEMF stimulation [PEMF(ON)] compared to various controls. A one-day stimulation protocol was used. Ctrl = non-stimulated group, [PEMF(OFF)] = apparatus turned off, sham = apparatus turned on without electromagnetic impulse. n = 5 independent experiments. Mean with standard deviation (SD). Non-stimulated group as control has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Gene Expression, Standard Deviation, Control

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Graphic representation of relative gene expression of complement factors (CD55, CD59, CFH and CFI) and cytokines (IL-6 and TNFα) in synovial fibroblast cell line K4IM after PEMF stimulation [PEMF(ON)] with and without 24 h TNFα pre-stimulation (10 ng/mL) compared to their respective control (ctrl). A one-day stimulation protocol was used. Ctrl = non-stimulation. n = 5 independent experiments. Mean with standard deviation (SD). Non-stimulated group without TNFα pre-stimulation (Ctrl/Ctrl) has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons (*). * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Gene Expression, Control, Standard Deviation

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Graphic representation of relative gene expression of complement factors (CD55, CD59, CFH and CFI) and cytokines (IL-6 and TNFα) in synovial fibroblast cell line K4IM after PEMF stimulation [PEMF(ON)] for 3 days and 6 days compared to their respective control (ctrl). Ctrl = non-stimulation. n = 4 independent experiments. Mean with standard deviation (SD). Non-stimulated group (3 days) has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons (*) * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Gene Expression, Control, Standard Deviation

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: ( A ) Representative images of K4IM cell line after 3 days and 6 days PEMF(ON) stimulation compared to their non-stimulated counterparts, respectively (200× magnification), immunolabeled with a CD59 specific antibody and negative control of the staining. Green (Alexa Fluor 488) = CD59, blue (DAPI) = cell nuclei, gray (Phalloidin Alexa Fluor 633) = F-Actin cytoskeleton. Scale bar = 100 µm. ( B ) Graphic representation of relative CD59 protein fluorescence intensity, n = 4 independent experiments, mean with standard deviation (SD). Control (3 days) has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Immunolabeling, Negative Control, Staining, Fluorescence, Standard Deviation, Control

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Antibodies and dyes used.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Concentration Assay

Journal: Cancer Biology & Therapy

Article Title: CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells

doi: 10.1080/15384047.2024.2314322

Figure Lengend Snippet: Flow cytometry analyses for CD38, CD46 and CD59. Daudi cells, SU-DHL-8, MOLP8, EJM, and MM.1 R cells were incubated with fluorophore-labeled anti-CD38, CD46 or CD59 antibodies. a) CD38. The gray peak in the flow histogram is the negative control (without antibody staining). The pink area represents cells stained with anti-CD38 antibody. b) CD46 and CD59. The gray curve is the negative control (without antibody staining). The blue curve represents cells detected by anti-CD46 or anti-CD59 antibodies. Shown is also the mean fluorescence intensity (MFI) of CD38, CD46 and CD59.

Article Snippet: The following antibodies were used: APC mouse anti-human CD46 (BD Biosciences), PE mouse anti-human CD59 (BD Biosciences), and PE anti-human CD38 (BioLegend).

Techniques: Flow Cytometry, Incubation, Labeling, Negative Control, Staining, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells

doi: 10.1080/15384047.2024.2314322

Figure Lengend Snippet: Co-treatment of MOLP8 cells with Ad35K++ and rILYd4overcomes resistance to isatuximab after the second cycle of treatment in MOLP8 and SU-DHL-8 cells. a) schematic of the experiment. Test cells (MOLP8 and SU-DHL8) were incubated with isatuximab/daratumumab in the presence of NHS to trigger CDC. CD46 and CD59 MFIs were measured before and 16 hours after adding mAb/NHS (c and f). Cell viability was counted 16 hours after mAb/NHS (b, e, h). Cells were then either incubated with or without Ad35K++/rILYd4 and isatuximab or daratumumab (d, g, i). (b-d) studies with MOLP8 cells. e-i) studies with SU-DHL-8 cells. For each data set, three independent experiments with three technical replicas were performed. *** p < .01, n.S. not significant.

Article Snippet: The following antibodies were used: APC mouse anti-human CD46 (BD Biosciences), PE mouse anti-human CD59 (BD Biosciences), and PE anti-human CD38 (BioLegend).

Techniques: Incubation

Journal: The Journal of Immunology Author Choice

Article Title: The Influence of Human IgG Subclass and Allotype on Complement Activation

doi: 10.4049/jimmunol.2300307

Figure Lengend Snippet: Complement activation of IgG1 and IgG3 is independent of complement regulators. Complement-dependent cytotoxicity (CDC) activity of IGHG1*03 (black), IGHG3*01 (long hinge, red), and IGHG3*04 (short hinge, pink) specific to trinitrophenyl (TNP) on Raji and Ramos B cells including CD46, CD55, and CD59 knockout cell lines at an Ag density of 0.5 mM (medium) TNBS. Negative controls are shown as dotted line for TNP unlabeled cells, and a dashed line represents TNPylated cells in the absence of Abs. Data represent mean ± SEM of n = 3.

Article Snippet: To study the expression of different complement regulators, conjugated Abs were tested at the following dilutions: mouse anti-human CD46-3–FITC (1:229; in-house mAb), mouse anti-human CD55-allophycocyanin (1:250; clone IA10; BD Biosciences), mouse anti-human CD59-FITC (1:100; clone MEM-443; Thermo Fisher Scientific), rabbit anti-human complement receptor 1 (CR1; 1:500; in-house mAb) in combination with goat anti–rabbit IgG Alexa Fluor 488 (1:500; clone A11034; Invitrogen) and mouse anti–human CR2-BV605 (1:100; clone BB-ly4; BD Biosciences).

Techniques: Activation Assay, Activity Assay, Knock-Out

Journal: Journal of neuroinflammation

Article Title: Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

doi: 10.1186/s12974-023-02920-9

Figure Lengend Snippet: Fig. 2 Localization of CD59 in murine teased nerve and myelinated cultures. A CD59 (green) and ankyrin-G (AnkG, red) staining of WT teased fibers. B CD59 (green) and Caspr (red) staining of WT teased fibers. C CD59 (green) and neurofascin (Nfasc, blue) staining of WT teased fibers. D CD59 (green), myelin basic protein (MBP, red), and neurofascin (Nfasc, blue) staining of WT ICR DRG. CD59 is co-localized with MBP and localized in the internodes and paranodes. There is no CD59 localization in the nodes of Ranvier. A–C Sections taken from a 4-month-old mouse. Immunolabeling procedure handled with methanol and 0.1% triton. Scale bars 20 μm and in D 50 μm

Article Snippet: Paraffin-embedded sections of human sural biopsies were stained using IHC methods for mouse mAb against human CD59 (BIO-RAD, Hercules, CA, USA) at 1:400 dilutions, rabbit mAb against human CD55 antibody (Abcam, CB, UK) at 1:400 dilution, rabbit mAb against human CD46 (Abcam, CB, UK) at 1:1000 dilution, mouse mAb against human CD35 (Abcam, CB, UK) at 1:100 dilution, rabbit pAb against human MBP (Zymed, Waltham, MA, USA), mouse mAb against human neurofilament (NF) (Dako, Glostrup South, Denmark), and rabbit pAb against human S-100 (Dako, Glostrup South, Denmark).

Techniques: Staining, Immunolabeling

Journal: Journal of neuroinflammation

Article Title: Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

doi: 10.1186/s12974-023-02920-9

Figure Lengend Snippet: Fig. 1 Localization of CD59 in peripheral nerve and murine sciatic nerve cross-sections. A–D CD59 (green), P0 (red) and beta dystroglycan (DG, blue) staining of murine wild-type (WT) and knock-out (KO) cross section fibers (A-D small squares). E–G CD59 (green) and myelin-associated glycoprotein (MAG, red) staining of fibers from WT mice. Staining shows that CD59 and P0, which represent compact myelin, have identical patterns in WT mice. Sections were taken from 4-month-old mice. Immunolabeling was performed with methanol and 0.1% triton. Scale bars 20 μm

Article Snippet: Paraffin-embedded sections of human sural biopsies were stained using IHC methods for mouse mAb against human CD59 (BIO-RAD, Hercules, CA, USA) at 1:400 dilutions, rabbit mAb against human CD55 antibody (Abcam, CB, UK) at 1:400 dilution, rabbit mAb against human CD46 (Abcam, CB, UK) at 1:1000 dilution, mouse mAb against human CD35 (Abcam, CB, UK) at 1:100 dilution, rabbit pAb against human MBP (Zymed, Waltham, MA, USA), mouse mAb against human neurofilament (NF) (Dako, Glostrup South, Denmark), and rabbit pAb against human S-100 (Dako, Glostrup South, Denmark).

Techniques: Staining, Knock-Out, Immunolabeling

Journal: Journal of neuroinflammation

Article Title: Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

doi: 10.1186/s12974-023-02920-9

Figure Lengend Snippet: Fig. 3 Localization of CD59 in human sural nerve. A–C Immunohistochemical staining for CD59 (DAB, red) in sural nerves of a healthy control. Healthy control staining shows compact myelin localization (C, blue arrow heads). CD59 was also detected in endothelial blood vessels of the epineurium (A and B, red arrow heads) and endoneurium (B and C, black arrow heads), as well as the perineurium (A and B, green arrow heads). D, E Immunofluorescence staining for CD59 (red) and MBP (blue). F, G Immunofluorescence staining for CD59 (red) and CD31 (green). Scale bars: A 500 μm, B 100 μm, C–G 50 μm. Magnifications: A ×4, B, D–G and ×20, C ×40. A–C 1:400 dilution

Article Snippet: Paraffin-embedded sections of human sural biopsies were stained using IHC methods for mouse mAb against human CD59 (BIO-RAD, Hercules, CA, USA) at 1:400 dilutions, rabbit mAb against human CD55 antibody (Abcam, CB, UK) at 1:400 dilution, rabbit mAb against human CD46 (Abcam, CB, UK) at 1:1000 dilution, mouse mAb against human CD35 (Abcam, CB, UK) at 1:100 dilution, rabbit pAb against human MBP (Zymed, Waltham, MA, USA), mouse mAb against human neurofilament (NF) (Dako, Glostrup South, Denmark), and rabbit pAb against human S-100 (Dako, Glostrup South, Denmark).

Techniques: Immunohistochemical staining, Staining, Control, Immunofluorescence

Journal: Journal of neuroinflammation

Article Title: Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

doi: 10.1186/s12974-023-02920-9

Figure Lengend Snippet: Fig. 4 Electron microscopy (EM) and immunofluorescence labeling of murine sciatic nerve. EM pictures of cross sections from WT (A–D (and CD59a-deficient (E–H) murine sciatic nerve at different ages. A and E are low-resolution toluidine blue pictures from a 6-month-old mouse. Scale bars: A and E 50 μm, B and F 10 μm, C, D, G and H 5 μm. I–L EM (higher magnification) showing accumulation of axoplasmatic organelles at an older age (18 months) in longitudinal sections from the paranodal regions (I, J, scale bars: I 2 μm, J 5 μm), and mitochondria and dense bodies in cross sections at regions with of noncompact myelin (K, scale bar = 500 nm; I–K are from CD59 KO mice). WT vs CD59 KO mice, comparison of the percentage of nodes of Ranvier with accumulation of axoplasmic organelles is shown in L (WT: n = 5, 82 nodes. KO: n = 5, 117 nodes; (p = 0.01, unpaired two-tailed student’s t-tests). M–P Immunofluorescence labeling of WT (left panels) and CD59 KO (right panels) teased fibers show normal node structure by CD59 (green) and AnkG (M, red), Caspr (N, red), neurofascin (O, Nfasc, blue), and myelin-associated glycoprotein (P, MAG, red). Sections taken from a 4-month-old mouse. Immunolabeling procedure handled with methanol and 0.1% triton. Scale bars 20 μm

Article Snippet: Paraffin-embedded sections of human sural biopsies were stained using IHC methods for mouse mAb against human CD59 (BIO-RAD, Hercules, CA, USA) at 1:400 dilutions, rabbit mAb against human CD55 antibody (Abcam, CB, UK) at 1:400 dilution, rabbit mAb against human CD46 (Abcam, CB, UK) at 1:1000 dilution, mouse mAb against human CD35 (Abcam, CB, UK) at 1:100 dilution, rabbit pAb against human MBP (Zymed, Waltham, MA, USA), mouse mAb against human neurofilament (NF) (Dako, Glostrup South, Denmark), and rabbit pAb against human S-100 (Dako, Glostrup South, Denmark).

Techniques: Electron Microscopy, Immunofluorescence, Labeling, Comparison, Two Tailed Test, Immunolabeling

Journal: Journal of neuroinflammation

Article Title: Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

doi: 10.1186/s12974-023-02920-9

Figure Lengend Snippet: Fig. 5 CD59 patient sural nerve biopsy. A, B Immunohistochemical staining of CD59 (DAB, red) from a healthy control subject (A) and a CD59 patient (B). CD59-deficient patient staining of CD59 was completely absent in comparison to a healthy control subject where CD59 appears to localize in compact myelin. Scale bars = 50 μm, magnifications × 40, 1:400 dilution. C–H Hematoxylin and eosin (H&E) (C and D), epoxy-resin embedded semi-thin sections stained with toluidine-blue (E and F), immunohistochemical staining for MBP (G) and neurofilament (NF) (H). Staining displayed normal nerve fiber density without evidence of active axonal or myelin damage. Scale bars: C, D, G and H 50 μm; E and F 20 μm; magnifications: C, D, G and H ×40; E and F ×60. I Electron microscopy image, tissue from a CD59-deficient patient showing thin myelin sheaths relative to axonal diameter with a relatively high g-ratio, suggestive of remyelination. Scale bar 5 μm. small squares, myelin sheath lamellar structure. Scale bar 500 nm

Article Snippet: Paraffin-embedded sections of human sural biopsies were stained using IHC methods for mouse mAb against human CD59 (BIO-RAD, Hercules, CA, USA) at 1:400 dilutions, rabbit mAb against human CD55 antibody (Abcam, CB, UK) at 1:400 dilution, rabbit mAb against human CD46 (Abcam, CB, UK) at 1:1000 dilution, mouse mAb against human CD35 (Abcam, CB, UK) at 1:100 dilution, rabbit pAb against human MBP (Zymed, Waltham, MA, USA), mouse mAb against human neurofilament (NF) (Dako, Glostrup South, Denmark), and rabbit pAb against human S-100 (Dako, Glostrup South, Denmark).

Techniques: Immunohistochemical staining, Staining, Control, Comparison, Electron Microscopy

Journal: Journal of neuroinflammation

Article Title: Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

doi: 10.1186/s12974-023-02920-9

Figure Lengend Snippet: Fig. 6 Localization of complement membrane regulatory proteins in murine teased fibers of sciatic nerve. Staining of WT teased fibers. CD55 (red), neurofascin (Nfasc, blue) and CD59 (green) (A), CD46 (red), neurofascin (Nfasc, blue) and CD59 (green) (B), Crry (red), neurofascin (Nfasc, blue) and CD59 (green) (C). Sections taken from a 4-month-old mouse. Immunolabeling procedure performed with the TSA method (A, C) and methanol and 0.1% triton (B). Scale bars 20 μm. CD55 staining was localized in Schmidt Lanterman incisures. CD46 staining was localized in the paranodes-juxtaparanodes, and Crry staining was localized in the paranodes and the internodes corresponding to compact myelin. White arrowheads indicate the nodes of Ranvier

Article Snippet: Paraffin-embedded sections of human sural biopsies were stained using IHC methods for mouse mAb against human CD59 (BIO-RAD, Hercules, CA, USA) at 1:400 dilutions, rabbit mAb against human CD55 antibody (Abcam, CB, UK) at 1:400 dilution, rabbit mAb against human CD46 (Abcam, CB, UK) at 1:1000 dilution, mouse mAb against human CD35 (Abcam, CB, UK) at 1:100 dilution, rabbit pAb against human MBP (Zymed, Waltham, MA, USA), mouse mAb against human neurofilament (NF) (Dako, Glostrup South, Denmark), and rabbit pAb against human S-100 (Dako, Glostrup South, Denmark).

Techniques: Membrane, Staining, Immunolabeling

Journal: Frontiers in Immunology

Article Title: Inhibition of complement activation by CD55 overexpression in human induced pluripotent stem cell derived kidney organoids

doi: 10.3389/fimmu.2022.1058763

Figure Lengend Snippet: iPSCs are vulnerable to complement activation. (A) HLA-ABC surface protein expression measured by flow cytometry in unstained (US), unstimulated (–) and IFN-γ stimulated (+) iPSCs. A representative histogram is shown of three independent experiments (n=3). (B) mRNA expression of CD46, CD55 and CD59 in iPSCs measured by RT-qPCR (n=3). (C) Complement inhibitor CD46, CD55 and CD59 protein surface expression in iPSCs measured by flow cytometry. Representative histograms are shown of three independent experiments (n=3). (D) Schematic of the experimental procedure to measure complement deposition on iPSCs in vitro . (E) C3 deposition on iPSCs incubated with heat-inactivated (dNHS) or active normal human serum (NHS) measured by flow cytometry. Results are presented as relative mean fluorescence intensity (MFI) and C3 + proportion. A representative histogram of three independent experiments (n=3) is shown and indicates the gate for the C3 + proportion. (F) C5b-9 deposition on iPSCs incubated with heat-inactivated (dNHS) or active normal human serum (NHS) measured by flow cytometry. Results are presented as relative mean fluorescence intensity (MFI) and C5b-9 + proportion. A representative histogram of three independent experiments (n=3) is shown and indicates the gate for the C5b-9 + proportion. Error bars show standard deviation and significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) was evaluated using T-test.

Article Snippet: For flow cytometry the following antibodies were used: PE-conjugated mouse anti- HLA-ABC (1:100, 555553, BD Biosciences), human anti- HLA-A2 antibody (3 µg mL -1 , SN607D8), mouse anti-human CD55 (1:500, ab1422, Abcam), mouse anti-C3 (1:1000, RFK22, in-house generated), mouse anti-C5b-9 (1:100, AE11, Hycult Biotech), PE-conjugated mouse anti-human CD46 (1:100, 12-0469-42, Invitrogen), and APC-conjugated mouse anti-human CD59 (1:100, 17-0596-42, Invitrogen) were used as primary antibodies.

Techniques: Activation Assay, Expressing, Flow Cytometry, Quantitative RT-PCR, In Vitro, Incubation, Fluorescence, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of complement activation by CD55 overexpression in human induced pluripotent stem cell derived kidney organoids

doi: 10.3389/fimmu.2022.1058763

Figure Lengend Snippet: CD55 overexpression in iPSCs decreases complement deposition. (A) Cell surface opsonization by anti-HLA-A2 in unstimulated (Unstim) and IFN- γ stimulated (Ctrl, GFP, CD55 #2, 4 and 6) iPSCs measured by flow cytometry. A representative histogram is shown and results are presented as mean fluorescence intensity (MFI) in three independent experiments (n=3). Staining with only PE-conjugated secondary antibody (2° ab) is shown in light grey. (B) CD46 and CD59 protein surface expression on iPSCs measured by flow cytometry (n=3). Staining with isotype (Iso) is shown in light grey. ( C, D ) C3 and C5b-9 deposition on iPSCs incubated with normal human serum (NHS) measured by flow cytometry. Incubation with heat-inactivated NHS (dNHS) was used as negative control and is shown in light grey. Results are presented as relative mean fluorescence intensity (MFI) and C3 + or C5b-9 + proportion. A representative histogram is shown (n=3 for control, n=5 for others). Error bars show standard deviation and significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) was evaluated using one-way ANOVA comparing each sample to iPSC-GFP with Dunnett correction for multiple comparisons. ns, non significant.

Article Snippet: For flow cytometry the following antibodies were used: PE-conjugated mouse anti- HLA-ABC (1:100, 555553, BD Biosciences), human anti- HLA-A2 antibody (3 µg mL -1 , SN607D8), mouse anti-human CD55 (1:500, ab1422, Abcam), mouse anti-C3 (1:1000, RFK22, in-house generated), mouse anti-C5b-9 (1:100, AE11, Hycult Biotech), PE-conjugated mouse anti-human CD46 (1:100, 12-0469-42, Invitrogen), and APC-conjugated mouse anti-human CD59 (1:100, 17-0596-42, Invitrogen) were used as primary antibodies.

Techniques: Over Expression, Flow Cytometry, Fluorescence, Staining, Expressing, Incubation, Negative Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Inhibition of complement activation by CD55 overexpression in human induced pluripotent stem cell derived kidney organoids

doi: 10.3389/fimmu.2022.1058763

Figure Lengend Snippet: CD55 overexpression in kidney organoids decreases complement deposition. (A) Representative fluorescent images of organoids with detection of CD55 (yellow) in high magnification. For the CD55 #2 organoid an image with detection of Hoechst (blue), NPHS1 (green), LTL (pink) and CD55 (yellow) of the same area as the separate CD55 image is included. Other clones are included in Supplemental Figure 4 . (B) CD55 mRNA expression in organoids at day 7 + 14 measured by RT-qPCR (n=3). (C) CD55 protein expression in dissociated kidney organoids at day 7 + 14 measured by flow cytometry showing a representative histogram of 5 independent experiments (n=5). Staining with only PE-conjugated secondary antibody (2° ab) is shown in light grey. (D) Cell surface opsonization by anti-HLA-A2 in unstimulated (Unstim) and IFN-γ stimulated (Ctrl, GFP, CD55 #2, 4 and 6) kidney organoid cells measured by flow cytometry. A representative histogram is shown of 5 independent experiments (n=5) and staining with only PE-conjugated secondary antibody (2° ab) is shown in light grey. (E) CD46 and CD59 protein surface expression on kidney organoid cells measured by flow cytometry (n=5). Staining with isotype (Iso) is shown in light grey. ( F , G ) C3 and C5b-9 deposition on kidney organoid cells incubated with normal human serum (NHS) measured by flow cytometry. Incubation with heat-inactivated NHS (dNHS) was used as negative control and is shown in light grey. Results are presented as C3 + or C5b-9 + proportion. A representative histogram is shown of 5 independent experiments (n=5). Error bars show standard deviation and significance (*p < 0.05; ***p < 0.001; ****p < 0.0001) was evaluated using one-way ANOVA comparing each sample to iPSC-GFP-derived kidney organoids with Dunnett correction for multiple comparisons. ns, non significant.

Article Snippet: For flow cytometry the following antibodies were used: PE-conjugated mouse anti- HLA-ABC (1:100, 555553, BD Biosciences), human anti- HLA-A2 antibody (3 µg mL -1 , SN607D8), mouse anti-human CD55 (1:500, ab1422, Abcam), mouse anti-C3 (1:1000, RFK22, in-house generated), mouse anti-C5b-9 (1:100, AE11, Hycult Biotech), PE-conjugated mouse anti-human CD46 (1:100, 12-0469-42, Invitrogen), and APC-conjugated mouse anti-human CD59 (1:100, 17-0596-42, Invitrogen) were used as primary antibodies.

Techniques: Over Expression, Clone Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Incubation, Negative Control, Standard Deviation, Derivative Assay