Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: ADR induces PLAD-dependent cleavage of DAF. (A and B) Representative images (A) and distribution of DAF expression (B) quantified in hiPod exposed to vehicle, ADR (0.3 µg/ml), PLADi (1 µM), or ADR + PLADi (0.3 µg/ml in 1 µM) for 24 h. DAF IF signal was normalized to actin expression pixel by pixel, and the MFI for each cell was computed. Results are representative of two independent experiments with similar results. (C and D) Representative blots (C) and densitometric analysis (D) of PLAD expression in hiPod cell lysates previously exposed to vehicle or ADR for 24 h. (E and F) Representative blots (E) and densitometric analysis (F) of DAF in the supernatants of hiPod exposed to ADR for 24 h with or without PLADi (WB). (G) Representative blot of DAF in the urine from BALB/c male mice at 2 wk after treatment with vehicle or ADR compared with recombinant mouse DAF (rDAF). In each group, we pooled and concentrated urine samples from eight mice (see Materials and methods). All experimental data were verified in at least three independent experiments. *P < 0.05; n.s., not significant. Scale bars: 50 µm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Expressing, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: FSGS in humans is associated with DAF down-regulation and complement activation. (A–D) C3 (A), C3aR (B), C5aR (C), and DAF mRNA (D) expression in glomeruli of human biopsy specimens with pathological diagnosis of FSGS or diabetic kidney disease compared with normal kidneys. Data are from previously published microarray studies by and were subjected to further analysis using Nephroseq. (E–H) Representative renal staining and data quantification for C3d (IF; E and F) and DAF (immunohistochemistry; G and H) in patients with FSGS ( n = 18) and in kidneys from healthy renal donors ( n = 10). (I) Correlation between protein and C3a in urine samples from 27 patients with FSGS taken at the time of kidney biopsy (before therapy). (J and K) Differences in proteinuria (J) and urinary C3a (K) measured before versus 3–6 mo after steroid therapy in a subset of 13 patients with FSGS. (L) Correlation between the change in proteinuria and change in urinary C3a before and after therapy for each of the same 13 patients. (M) Representative blot of DAF in the urine from healthy control individuals and patients with FSGS compared with recombinant human DAF (rDAF). In each group, we pooled and concentrated urine samples from five and five subjects, respectively (see Materials and methods). *P ≤ 0.05. Scale bars: 25 μm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Activation Assay, Expressing, Biomarker Discovery, Microarray, Staining, Immunohistochemistry, Control, Recombinant

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1 .

Article Snippet: Anti-human CD55 (clone BRIC216) , Bio-Rad , Cat: MCA914T; RRID: AB_1102203.

Techniques: CRISPR, Activation Assay, Transduction, Genome Wide, Staining, Stable Transfection, Expressing, Control, Flow Cytometry, In Vitro, Immunoprecipitation, Recombinant, Knock-Out

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet: Interaction of CD55 with HLA-C∗07:01-VRIG tetramers is allotype and peptide specific (A) HEK293T cells were transfected with a plasmid containing GFP and a truncation mutant of CD55 and analyzed by flow cytometry. GFP+ positive cells were analyzed for staining with HLA-C∗07:01-VRIG. Each mutant removes an additional SCR domain from CD55. Data are represented as mean ± SD. (B) HeLa cells were stained with HLA-C∗07:01-VRIG tetramers after pre-incubation with CD55 blocking antibodies targeting different SCR domains on CD55 and analyzed by flow cytometry. (C) HeLa cells were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with the VRIG peptide and analyzed by flow cytometry. (D) HeLa cells were stained with HLA-C∗07:01 tetramers loaded with different alanine mutants of the VRIGHLYIL peptide and analyzed by flow cytometry. (E) CD55-Fc was immobilized on a Prot-G chip for SPR data using HLA-C∗07:01-VRIG tetramers as analyte to determine interaction on and off rates and K D . Response units were measured with increasing concentrations of HLA-C∗07:01-VRIG tetramers. All data represent at least three independent experiments, except (E), which represents a biological duplicate. FL, full length. Related to Figure S2 , Tables S2 and .

Article Snippet: Anti-human CD55 (clone BRIC216) , Bio-Rad , Cat: MCA914T; RRID: AB_1102203.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Staining, Incubation, Blocking Assay

Journal: iScience

Article Title: CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

doi: 10.1016/j.isci.2024.110120

Figure Lengend Snippet:

Article Snippet: Anti-human CD55 (clone BRIC216) , Bio-Rad , Cat: MCA914T; RRID: AB_1102203.

Techniques: Virus, Recombinant, Blocking Assay, Genome Wide, Activation Assay, CRISPR, Knock-Out, Mutagenesis, Plasmid Preparation, Software, Imaging

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Gene identity, NCBI accession number, and primer sequences of the primers used for the real time PCR.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Amplification

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Levels of complement-regulatory protein CD55 remain unaltered, but CD46 levels decline in HeLa cells infected with vesicular stomatitis virus. ( A – G ) Whole cell lysates collected from mock- and VSV-infected HeLa cells at the indicated time points were subjected to immunoblotting to determine the expression of CD46 and CD55. Samples at the time point 0–3 h ( A ), 4–7 h ( B ), 8–11 h ( C ), 12–15 h ( D ), 16–18 h ( E ), 19–21 h ( F ) and 22–24 h ( G ), respectively. Anti-CD55 and CD46 antibodies were used to detect the levels of the corresponding proteins in the lysate at different time points. Virus infectivity was detected using a VSV anti-M antibody while actin served as the loading control. The levels of CD55 were maintained at all of the time points tested compared to the mock; however, the levels of CD46 declined significantly, starting from 15 h onwards (note the decreasing levels of CD46 in D , E , and the complete absence in F , G ). The entire panel of blots is representative of three independent experiments. ( H ) The densitometry analysis of the CD55 and CD46 protein expression, normalized against the loading control. The data represents the mean + SEM of the three independent experiments.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Infection, Western Blot, Expressing

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: VSV infection causes a decline in the surface expression of CD46 and not CD55. HeLa cells were infected with VSV (10 MOI) for the specified time points. The surface distribution of CD55 and CD46 was determined by staining the mock- and VSV-infected cells with anti-CD55 and CD46 primary antibodies, and by counter staining with AF488-labelled secondary antibody. The two panels, A and B , denote the histogram representing the fluorescence intensity on the x -axis and the cell count on the y -axis. The infection of the HeLa cells with VSV did not alter the surface level expression of CD55 ( A ) even until 24 h; however, a drastic reduction in the surface expression of CD46 ( B ) could be evidenced 6 h post infection.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Infection, Expressing, Staining, Fluorescence, Cell Counting

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Vesicular stomatitis virus infection leads to down-regulation of CD55 and CD46 transcripts. A comparative analysis of the relative levels of CD55 ( A ) and CD46 ( B ) mRNA in VSV and mock-infected HeLa cells at 6, 12, 18 and 24 h was carried out by RT-qPCR. The total RNA isolated from the mock- and VSV-infected cells was converted to cDNA. Equal concentrations of cDNA from all of the samples were used to analyze the gene expression at various time points post VSV-infection using Taqman gene expression assays. The fold change was calculated by the comparative Ct method (2^- ddCt). The statistical significance was calculated using Students t -test, with * p ≤ 0.01; ** p ≤ 0.001; **** p ≤ 0.0001, and ns = non-significance.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Infection, Quantitative RT-PCR, Isolation, Expressing

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Cycloheximide chase assay to measure protein stability. ( A ) The HeLa cells were treated with Cycloheximide for the indicated times, and the whole cell lysate was subjected to immunoblotting in order to determine the stability of CD55 and CD46. β-actin was used as the loading control, and P53 served as the positive control. ( B ) Quantification of the immunoblot results by Image J software. The result represented is the average + SEM of four independent experiments obtained by normalizing the band intensity of the RCA against b-actin. The statistical significance was calculated using Student’s t -test.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Western Blot, Positive Control, Software

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Complement regulator CD55 is found in greater abundance than CD46 on VSV from HeLa cells. ( A ) Equal concentrations of protein in the purified virus (5 μg) were separated by SDS-PAGE and subjected to Western blotting. The proteins that have been probed are indicated in the right, and their corresponding molecular weights are in parentheses. ( B ) An ELISA specific to VSV was performed by coating wells with serially-diluted gradient-purified VSV. The adsorbed virus particles were detected using an anti-VSV-G antibody. Variability in the absorbance was observed even at similar concentration of viruses purified at varying time intervals. Across the samples, an absorbance of ~1.3 was found to be common; this is indicated by the lines drawn against the optical density. ( y -axis) to the corresponding concentrations ( x -axis).

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Purification, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: Levels of CD55 and CD46 associated with sucrose gradient-purified VSV from HeLa cells at various time intervals. a,b The concentration of the virion-associated CD55/CD46 was calculated from the pixel intensity of the known concentrations of rCD46 and rCD55 run in parallel ( Figure 5 A). The concentration of CD55 and CD46 is depicted as ng/5μg of VSV. c,f Based on the densitometric analysis of VSV-G or M, further normalization was performed relative to the values obtained at 6–12 h (taken as 1). The values indicate the decline in the concentration of G or M relative to 6–12 h. d,g The ratio of CD55 to VSV-G or VSV-M was calculated by dividing the CD55 concentration at a specific time point by the normalized levels of G or M. The values in d were obtained by dividing a by c , and those in e by dividing b by c . Similarly, the ratios in g and h were obtained by dividing a by f and b by f , respectively.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Concentration Assay

Journal: Viruses

Article Title: Functional Dissection of the Dominant Role of CD55 in Protecting Vesicular Stomatitis Virus against Complement-Mediated Neutralization

doi: 10.3390/v13030373

Figure Lengend Snippet: CD55 confers greater resistance to VSV against complement compared to CD46. ( A ) Western blot depicting the level of expression of CD55 and CD46 in HeLa and A549 cells; β-actin served as the equal loading control. ( B ) The effect of NHS in neutralizing VSV grown in HeLa and A549 cells was assessed by a plaque reduction assay. The virus harvested at the indicated time intervals was incubated either with NHS or PBS (black bars). At all of the time points tested, the HeLa-grown viruses showed marked resistance to complement-mediated neutralization. The degree of neutralization of A549-grown VSV known to harbor less CD55 was significantly higher than that of HeLa-grown VSV at 12–18 h ( p < 0.0001). The symbols in the graph represent * p < 0.05; ** p < 0.005; *** p < 0.0005). The additional symbols represent the comparison of significance between A549- and HeLa-grown VSV treated with NHS at the respective time ranges, where # p < 0.0005; $ p < 0.0005; ^ p < 0.0001; @ p < 0.005.

Article Snippet: The rabbit and mouse monoclonal antibodies against CD55 and CD46 used in flow cytometry experiments, and the recombinant CD55 and CD46 were sourced from Sino Biological Inc. (Beijing, China).

Techniques: Western Blot, Expressing, Incubation, Neutralization

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Expression of CD55 and CD59 in tumor cells. (A) Cell lines were incubated with fluorescein isothiocyanate-conjugated anti-human CD55 and CD59 monoclonal antibodies, and analyzed by flow cytometry. Error bars depict standard deviations. (B) The protein level of CD55 and CD59 in A549 and Lovo cells were detected by western blotting. β-actin protein levels served as the loading control. CD55, decay accelerating factor; CD59, protectin.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Expressing, Incubation, Bioprocessing, Flow Cytometry, Western Blot, Control

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Establishing stable transfected α-gal-expressing cell lines. (A) α-1,3GT mRNA expression in A549, A549-V, A549-GT, Lovo, Lovo-V and Lovo-GT cells were detected by reverse transcription-polymerase chain reaction. The amplified product of α-1,3GT was detected by agarose gel electrophoresis in lane 2, 4 and 6 of the two gels. GAPDH was used as loading control in lane 1, 3 and 5 of the two gels. (B) Expression of α-gal epitope in each group of cells were detected by direct immunofluorescence (magnification, ×200). FITC-conjugated BS-IB4 lectin staining was performed to probe α-gal epitope. (B-a) A549-GT, (B-b) A549, (B-c) A549-V, (B-d) Lovo-GT, (B-e) Lovo, (B-f) Lovo-V and (B-g) positive control PIEC cells. (C) Expression of α-gal epitope in each group of cells were stained with FITC-BS-IB4 lectin, then analyzed by flow cytometry. Error bars depict standard deviations. FITC, fluorescein isothiocyanate; α-gal, Galα1-3Galβ1-4GlcNAc-R; α-1,3GT, α1,3-galactosyltransferase; PIEC, pig iliac arterial endothelial cells; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Transfection, Expressing, Reverse Transcription, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Control, Immunofluorescence, Staining, Positive Control, Flow Cytometry

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Expression of CD55 and CD59 on α-gal-expressing cells influences their sensitivity to CDC. (A) A549, A549-V, A549-GT, Lovo, Lovo-V, Lovo-GT and positive control PIEC cells were incubated with various dilutions of NHS (0, 15, 30, 50%) and survival rates were analyzed by trypan blue staining. Error bars showed standard deviations (*P<0.05 vs. the control). (B) A549, A549-V, A549-GT Cells were pre-treated with various concentrations of PI-PLC (0.001, 0.01, 0.05, 0.1, 0.2, or 0.5 U/ml), incubated with 50% NHS, and survival rates were analyzed by trypan blue staining. Error bars showed standard deviations. *P<0.05, vs. the control. CD55, decay accelerating factor; CD59, protectin; NHS, normal human serum; PI-PLC, phosphatidylinositol-specific phospholipase C; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Expressing, Positive Control, Incubation, Staining, Control

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Effects of PI-PLC treatment on CD55 and CD59 protein level in A549-GT cells. (A) Following 0.1 U/ml PI-PLC treatment, CD55 and CD59 were tested by western blot in A549, A549-V, A549-GT, Lovo, Lovo-V and Lovo-GT cells, compared with that prior to PI-PLC treatment. (B) After 0.1 U/ml PI-PLC treatment, A549-GT cells was incubated with fluorescein isothiocyanate-conjugated anti-human monoclonal antibodies. CD55 and CD59 were analyzed by flow cytometry, compared with that prior to PI-PLC treatment. Error bars showed standard deviations. *P<0.05 vs. the control. CD55, decay accelerating factor; CD59, protectin; PI-PLC, phosphatidylinositol-specific phospholipase C; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Western Blot, Incubation, Bioprocessing, Flow Cytometry, Control, Expressing

Journal: Oncology Letters

Article Title: Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen

doi: 10.3892/ol.2018.8478

Figure Lengend Snippet: Effect of anti-CD55 and anti-CD59 on CDC in α-gal-expressing cells. The A549, A549-V and A549-GT cells were pre-incubated with each antibodies (anti-CD55, anti-CD59 and anti-CD55 with anti-CD59) (10 µg/ml), then 50% normal human serum was added and the survival rates were calculated. Error bars showed standard deviations. *P<0.05 vs. the control. CD55, decay accelerating factor; CD59, protectin; α-gal, Galα1-3Galβ1-4GlcNAc-R; A549-GT, α-gal expressing A549; A549-V, control.

Article Snippet: Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD55 (cat. no. FHF055) and CD59 (cat. no. FHF059) mAbs were purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China).

Techniques: Expressing, Incubation, Control

Journal:

Article Title: Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-?(1-3) Galactosyl Epitope

doi: 10.1128/JVI.78.11.5812-5819.2004

Figure Lengend Snippet: hCD55 and α-Gal expression on porcine cells. (a) Expression of CD55 was analyzed by FACs on both transgenic (TgPAE) and nontransgenic (PAE) pig cells. Staining by anti-hCD55 (shaded histogram), no primary antibody (negative control; solid line), or anti-hCD46 (dotted line, similar profile to that of the no-antibody control) followed by FITC-labeled secondary anti-mouse immunoglobulin antibodies (upper panels) is shown. Staining for α-Gal was a one-step incubation; hence, −lectin on the histogram represents cells only, and +lectin represents cells incubated with IB-4 lectin conjugated to FITC (lower panels). (b) α-Gal expression on ST-IOWA wild-type and ST-IOWA Gal-null (−/−) cells. Expression of the α-Gal antigen was analyzed by FACs, using the IB-4 lectin. A rightward shift of the histogram in the presence of lectin (+lectin) compared to the histogram generated in the absence of lectin (−lectin) indicates expression of the α-Gal antigen. The level of expression was determined using the MFI shift, generated by the Cell Quest FACScan software. The MFI shift was calculated by determining test MFI and subtracting that of negative controls (no primary antibodies for hCD55 and hCD46 expression and no lectin for α-Gal expression), and all the mean shifts are shown in Table ​Table11.

Article Snippet: Samples were then incubated on ice with either primary mouse anti-human CD55 antibody (BRIC110) or mouse anti-human CD46 antibody (J4-48) from Cymbus Bioscience Ltd., diluted to 1:20 in PBS/BA, for 1 h. After three washes in PBS/BA, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), diluted to 1:200 in PBS/BA and incubated on ice for 1 h. For α-Gal expression, cells were detached by a cell scraper, washed, resuspended, and stained in a single 1-h incubation with FITC-conjugated Bandeiraea simplicifolia isolectin (IB-4) (Sigma) at 10 μg/ml in PBS/BA on ice, as this lectin is specific for the terminal α-Gal sugar.

Techniques: Expressing, Transgenic Assay, Staining, Negative Control, Labeling, Incubation, Generated, Software

Journal:

Article Title: Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-?(1-3) Galactosyl Epitope

doi: 10.1128/JVI.78.11.5812-5819.2004

Figure Lengend Snippet: Summary of virus sensitivity and cell surface molecule expression

Article Snippet: Samples were then incubated on ice with either primary mouse anti-human CD55 antibody (BRIC110) or mouse anti-human CD46 antibody (J4-48) from Cymbus Bioscience Ltd., diluted to 1:20 in PBS/BA, for 1 h. After three washes in PBS/BA, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), diluted to 1:200 in PBS/BA and incubated on ice for 1 h. For α-Gal expression, cells were detached by a cell scraper, washed, resuspended, and stained in a single 1-h incubation with FITC-conjugated Bandeiraea simplicifolia isolectin (IB-4) (Sigma) at 10 μg/ml in PBS/BA on ice, as this lectin is specific for the terminal α-Gal sugar.

Techniques: Expressing

Journal:

Article Title: Reduced Sensitivity to Human Serum Inactivation of Enveloped Viruses Produced by Pig Cells Transgenic for Human CD55 or Deficient for the Galactosyl-?(1-3) Galactosyl Epitope

doi: 10.1128/JVI.78.11.5812-5819.2004

Figure Lengend Snippet: Demonstration of hCD55 incorporation on VSV particles by a viral pull-down assay. VSV harvested through HeLa, TgPAE A, and PAE E cells in the presence of antibody was incubated with protein G-expressing bacterial cells (OMNISORB). Antibodies used were three anti-human CD55 antibodies, BRIC 216, BRIC 471, and anti-DAF; anti-human CD46 J4-48 (hCD46); and anti-CD59 BRA-10G (hCD59). Five micrograms of anti-DAF and 1 μg of the other antibodies were used for incubation with virus and protein G cells. Titers of VSV for pulled-down and input particles were determined by TCID50 assay. The ratio of these titers is shown as percent binding.

Article Snippet: Samples were then incubated on ice with either primary mouse anti-human CD55 antibody (BRIC110) or mouse anti-human CD46 antibody (J4-48) from Cymbus Bioscience Ltd., diluted to 1:20 in PBS/BA, for 1 h. After three washes in PBS/BA, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch), diluted to 1:200 in PBS/BA and incubated on ice for 1 h. For α-Gal expression, cells were detached by a cell scraper, washed, resuspended, and stained in a single 1-h incubation with FITC-conjugated Bandeiraea simplicifolia isolectin (IB-4) (Sigma) at 10 μg/ml in PBS/BA on ice, as this lectin is specific for the terminal α-Gal sugar.

Techniques: Pull Down Assay, Incubation, Expressing, TCID50 Assay, Binding Assay