alexa-fluor mab labeling kits  (Thermo Fisher)

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A) Neutralization of live EBOV, SUDV, or BDBV by the indicated monoclonal antibodies (mAbs) was assessed by plaque assay. Left: Neutralization of EBOV by varying concentrations of each mAb. Right: Summary table showing the 50 or 80% plaque reduction neutralization test (PRNT50 or PRNT80) for each mAb against the indicated viruses. (B) Grouping of the neutralizing mAbs into competition groups. The numbers in the table represent the binding of fluorescently labeled mAbs (columns) to EBOV GP-expressing cells in the presence of excess unlabeled competitor mAbs (rows). Binding in the presence of each competitor is expressed as a percentage of the fluorescence signal in the absence of competitor. mAbs were classified as soluble glycoprotein (sGP)-binding (+) or non-binding (−) by enzyme-linked immunosorbent assay (ELISA). mAb 9.20.1C3 was unique within the chalice bowl competition group in being unable to bind to sGP (yellow highlight).

Article Snippet: Briefly, Jurkat GP cells were pre-incubated with 100 μg/mL unlabeled competitor mAb on ice, then test mAbs labeled with the Alexa 488 protein labelling kit (ThermoFisher) were added at 2 μg/mL.

Techniques: Neutralization, Plaque Assay, Plaque Reduction Neutralization Test, Binding Assay, Labeling, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A–C) or surface representation (D, F). 1C11 single-chain variable fragment (scFv) is shown in blue, and 1C3 antigen-binding fragment (Fab) is shown in orange. The international immunogenetics information system (IMGT) numbering scheme is used for mAbs 1C11 and 1C3. (A) Closeup of 1C11-fusion loop interaction with key side chains of 1C11 (blue) and GP (grey) illustrated. GP residues that are conserved among five ebolaviruses (EBOV, SUDV, BDBV, RAFV, and RESTV) are underlined. (B) 1C11 draws the fusion loop away from the core of GP upon binding. The conformation of the fusion loop in an unbound GP (PDB:5JQ3) is shown in green, and the 1C11-bound conformation is shown in black. (C) The N-linked glycan at position 563 and the 1C11 complementarity determining region (CDR) side chains that interact with it are illustrated. Side chains within hydrogen bonding range to the glycan are indicated by dashed yellow lines. (D) 1C3 and the head region of EBOV GP are shown in closeup, including the long CDRL1 loop of 1C3 which extends deep into the EBOV GP “chalice”. (E) The footprints of 1C3 and 1C11 are highlighted on the sequence alignment of five major ebolaviruses. Residues were labeled by colored triangles at the bottom of the alignment to show the conservation (blue - conserved, pink - similar, yellow - non-conserved). The footprint of 1C3 contains five non-conserved residues, and the footprint of 1C11 contains only conserved or highly similar residues. (F) On the left, EBOV GP surfaces that directly contact 1C3 are shown in various shades of orange as follows: 1C3-bound residues in common with all three GP protomers are colored in dark orange, residues bound by only two protomers (A and B) are colored in medium orange, and residues unique to a single protomer are colored in light orange and yellow. On the right, the three separate portions of the tripartite 1C3 footprint on the GP protomers A, B, and C are illustrated. For example, residues 114–120 on protomer A are bound by CDRs H2 and H3, while the same residues 114–210 on protomer B are bound by 1C3 CDRL2 and framework region 3. CDRL1 of 1C3 simultaneously contacts residues 124–126 on both protomers A and B.

Article Snippet: Briefly, Jurkat GP cells were pre-incubated with 100 μg/mL unlabeled competitor mAb on ice, then test mAbs labeled with the Alexa 488 protein labelling kit (ThermoFisher) were added at 2 μg/mL.

Techniques: Binding Assay, Sequencing, Labeling

Journal: Cell

Article Title: Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses

doi: 10.1016/j.cell.2022.02.023

Figure Lengend Snippet: (A) Survival of mice treated with 1C3 or 1C11 prior to Ebola virus (EBOV) exposure. mAbs were administered 24 h prior to exposure with 100 plaque-forming units (PFU) of mouse-adapted EBOV. A human immunoglobulin G against influenza A virus (IgG1) mAb was used as a control. Animal survival was assessed twice daily for 21 d. n = 10 mice were studied per treatment condition. (B) Survival of STAT1 KO mice treated with 1C3 or 1C11 after EBOV/BDBV-GP exposure. Groups of STAT1 KO mice at five animals per group were injected with the indicated mAbs by the intraperitoneal route at 24 h after BDBV chimeric virus challenge. Kaplan-Meier survival curve is shown. Each group was compared with phosphate-buffered saline (PBS) control (Mantel-Cox test). (C and D) Groups of guinea pigs at five animals per group were injected with indicated mAbs by the intraperitoneal route at 1 or 3 days after EBOV (C) or Sudan virus (SUDV) (D) exposure. Kaplan-Meier survival curves, body weights, illness score curves, and viremia levels are shown. For analysis of survival, each group was compared to PBS mock control (Mantel-Cox test). In panels representing viremia, each dot corresponds to an individual serum sample. Short horizontal lines indicate the mean value of titers. The dotted horizontal lines show the detection limit. For analysis of viremia data, serum samples collected on different days were pooled together in each experimental group. Samples without detectable virus were arbitrarily assigned the viremia level values corresponding to the detection limit (102 PFU/mL). One-way analysis of variance (ANOVA) with Dunnett correction was used for multiple comparisons between each group and PBS mock control.

Article Snippet: Briefly, Jurkat GP cells were pre-incubated with 100 μg/mL unlabeled competitor mAb on ice, then test mAbs labeled with the Alexa 488 protein labelling kit (ThermoFisher) were added at 2 μg/mL.

Techniques: Injection

Journal: PLoS ONE

Article Title: Modulation of the Host Interferon Response and ISGylation Pathway by B. pertussis Filamentous Hemagglutinin

doi: 10.1371/journal.pone.0027535

Figure Lengend Snippet: PBMCs were separated into monocytes (Mono), lymphocytes (Lympho), and an NK-enriched cell population (NK), using magnetic beads. (A) For ISG15 mRNA expression, 2×10 6 cells/ml were cultured for 4, 8, and 20 hrs in the presence of IFN-α (IFN-α-treated cells) at 1,000 U/ml or FHA-2 (FHA-treated cells) at 5 µg/ml or without treatment. At each time point, total RNA was extracted and analyzed by RT-PCR. The fold expression change shown in the Figure is relative to levels in untreated cells at the beginning of the time course; the standard deviations for duplicate measurements are displayed. (B) For intracellular detection of ISG15, monocytes, NK-enriched cells, and lymphocytes were stimulated for 4 hrs with IFN-α (IFN-α-treated cells) at 1,000 U/ml or FHA-2 (FHA-treated cells) at 5 µg/ml or without treatment. Cells were then stained for intracellular ISG15 using the mouse monoclonal anti-ISG15 clone 4.1 labeled with AlexaFluor 647 (AF647). The number in the upper left corner of each graph represents the percentage of total cells testing positive for ISG15.

Article Snippet: Alexa Fluor 647-conjugated antibodies were obtained by labelling 100 µg of mouse monoclonal anti-ISG15 antibody (clone 4.1, a generous gift from Dr. E. Borden, Cleveland Clinic Foundation, Cleveland, OH) and 100 µg of purified mouse IgG1 (eBioscience, San Diego, CA) using the Alexa Fluor 647 monoclonal antibody labelling kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions.

Techniques: Magnetic Beads, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Staining, Labeling

Journal: PLoS ONE

Article Title: DEspR Roles in Tumor Vasculo-Angiogenesis, Invasiveness, CSC-Survival and Anoikis Resistance: A ‘Common Receptor Coordinator’ Paradigm

doi: 10.1371/journal.pone.0085821

Figure Lengend Snippet: (A) Merged immunofluorescence image of Gr. IV PDAC tumor tissue showing DEspR+ expression (red) in PDAC ductal tumor cells (white arrows) and alpha smooth muscle actin (aSMA)+ expression (green) in adjacent stromal tissue (upper left). DEspR+ and aSMA+ expression and co-expression (yellow) in PDAC tumor microvessels (lower left). Merged immunofluorescence images of normal pancreas detects no DEspR expression in ducts, parenchymal cells (upper right) nor in microvessels (white arrows, lower right). Differential interference contrast (DIC) overlays showing clusters of DEspR+ (red) microvessels (yellow arrows) and co-expressed DEspR+/aSMA+ (yellow) in microvessels (white arrows) within stromal fibrosis area (lower left) in pancreatic cancer but none in normal tissue. (B) Individual and merged immunofluorescence images of DEspR (red), CD133 (green) and double DEspR+/CD133+ immunofluorescence (yellow) shows DEspR+,CD133+ co-expression (yellow) in pancreatic cancer cells at invasive tumor edge (white arrows). Normal pancreatic tissue does not exhibit DEspR+ and CD133+ immunostaining. (C) DEspR+ (red) GBM tumor cells and microvascular endothelium at tumor edge with CD133+ (green) putative CSCs at GBM tumor edge. Merged DEspR+/CD133+ co-expression (yellow-green or yellow) in GBM putative CSCs (white arrows) close to DEspR+ microvessels (yellow arrows). AF568-labeled isotype control (Isotype) shows negative immunofluorescence of GBM section at identical experimental settings; microvessel (yellow arrow). No DEspR+ expression detected in normal brain section. Tumor biopsy core analysis in . Bar, 20 microns.

Article Snippet: 7c5b2 mAb was labeled with Alexa Flour 568 using the Alexa Fluor 568 Monoclonal Antibody Labeling Kit (Invitrogen).

Techniques: Immunofluorescence, Expressing, Immunostaining, Labeling

Journal: PLoS ONE

Article Title: DEspR Roles in Tumor Vasculo-Angiogenesis, Invasiveness, CSC-Survival and Anoikis Resistance: A ‘Common Receptor Coordinator’ Paradigm

doi: 10.1371/journal.pone.0085821

Figure Lengend Snippet: (A) FACS analysis of anchorage-independent Panc1-CSCs and U87-CSCs comparing control double isotype immunophenotyping (IgG2b-AF568 for 7c5b2 and IgG1-AF488 for CD133) with double anti-DEspR-AF568 and anti-CD133-AF488 immunophenotyping. (B) B-i. ARMS assay specific for spliced DEspR-RNA 270-bp amplicon (red arrow) spanning the spliced exon-to-exon junction ( ,C) confirms presence of spliced DEspR-RNA in Panc1 (lane 1) and U87 (lane 2) CSCs. B-ii. Quantitative RT-PCR Ct plot. B-iii. Corresponding agarose-gel size fractionation of RT-PCR products demonstrating expected-size DEspR-specific 88-bp amplicon, and control cyclophilin-specific 145-bp amplicon band. DNA size standards: 50-bp DNA ladder). B-iv. Calculation of qRT-PCR ΔCt fold-change in both Panc1- and U87-anchorage independent CSCs. B-v. Western blot analysis detects predicted ∼10 kDa DEspR protein (red arrow) in membrane proteins isolated from U87 CSCs in three independent experimental conditions: 1X, 1∶100 anti-DEspR ab-dilution in two different detection systems; 2X, 1∶50 dilution of anti-DEspR mAb in ECL detection system. (C) Analysis of survival in adverse conditions comparing Panc1- (red open circle) and U87- (blue open square) nonCSCs with quinary CSCs: Panc1 (red circle) and U87 (blue square). Analysis of effects of anti-DEspR (7c5b2) treatment on U87-CSC survival measured as %-live CSCs comparing quinary 7c5b2-treated U87-CSCs (black open circle) and control non-treated U87-CSCs (black circle) in identical adverse conditions. (D) Analysis of effects of DEspR-inhibition on Panc1- and U87-CSC survival in suspension culture at physiological conditions measured as number of live cells and % dead cells comparing quinary anchorage-independent 7c5b2-treated CSCs (black bars) and control non-treated CSCs (white bars). ANOVA P<0.0001, Tukey's multiple pairwise comparisons tests: ***, P<0.0001; **, P<0.001; *, P<0.05.

Article Snippet: 7c5b2 mAb was labeled with Alexa Flour 568 using the Alexa Fluor 568 Monoclonal Antibody Labeling Kit (Invitrogen).

Techniques: Amplification, Quantitative RT-PCR, Agarose Gel Electrophoresis, Fractionation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Inhibition