Journal: Infection and Immunity

Article Title: Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae : cross-protective responses elicited by the 4CMenB meningococcal vaccine

doi: 10.1128/iai.00309-23

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: TABLE 1 Target Antibody/clone name Species, isotype Label a Concentration (mg/ml) Source Use a Comments 2C7 Anti- N. gonorrhoeae LOS IgG3 (-) 0.28 mg/ml S. Ram, UMass IFC, C3 deposition Hybridoma supernatant 6B4 Anti- N. gonorrhoeae LOS IgM, κ (-) 0.33 mg/ml S. Ram, UMass IFC, SBA Hybridoma supernatant CD11b, human M1/70 Rat IgG2b, κ PE 0.2 mg/ml Biolegend Flow cytometry Catalog # 101207 N. gonorrhoeae Rabbit (-) 4-5 mg/ml Meridian IFC, SBA, OPKA Catalog #B65111R IgG, rabbit AffiniPure, Fc-fragment specific Goat AF488 1.5 mg/ml Jackson Immuno Research IFC Catalog # 111-545-008 IgG, mouse Fcy fragment specific Goat AF488 1.5 mg/ml Jackson Immuno Research IFC Catalog # 115-545-164 IgM, mouse μ-Chain specific Goat AF647 1.4 mg/ml Jackson Immuno Research IFC Catalog # 115-606-075 C3, human Goat IgG FITC 7 mg/ml MP Biomedical IFC Catalog # 55167 Isotype control for mouse IgG E1D5H Mouse IgG3 FITC 2.5 mg/ml Cell Signaling Technology IFC Catalog # 37988 Isotype control for mouse IgM Clone MM-30 Mouse IgMκ APC 2-3 mg/ml Biolegend IFC Catalog # 401630 Isotype control for goat anti-human C3 Goat IgG (-) 1 mg/ml R&D Systems IFC Catalog # AB-108-C Open in a separate window a AF, AlexaFluor; APC, allophycocyanin; FITC, fluorescein isothiocyanate; IFC, imaging flow cytometry; OPKA, opsonophagocytic killing; PE, phycoerythrin; SBA, serum bactericidal activity.

Techniques: Concentration Assay, Flow Cytometry, Control

Journal: Infection and Immunity

Article Title: Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae : cross-protective responses elicited by the 4CMenB meningococcal vaccine

doi: 10.1128/iai.00309-23

Figure Lengend Snippet: Antibody binding to the surface of N. gonorrhoeae by imaging flow cytometry. (A) Gating strategy. N. gonorrhoeae strain FA1090 was incubated with rabbit anti-N. gonorrhoeae antibody and AlexaFluor 488 (AF488)-coupled anti-rabbit antibody, fixed, and incubated with DAPI. On the ImagestreamX imaging flow cytometer, samples were gated on (I) Root mean square (RMS) of >52, then (ii) focused bacteria, and then (iii) DAPI+ singlets, from which AF488-positive fluorescence was measured (iv). (B) Representative examples of individual N. gonorrhoeae of strain FA1090 that bind mouse (Ms) 2C7 anti-lipooligosaccharide IgG3, polyclonal rabbit (Rb) anti-N. gonorrhoeae IgG (αGc), and mouse 6B4 anti-lipooligosaccharide IgM (left) but not corresponding isotype controls (right). Number in the upper left corner of each phase image indicates the identity of the particle captured by the imaging flow cytometer. Measurements of mean fluorescence intensity (MFI) (C, F, I), percent-positive bacteria (D, G, J), and fluorescence index (FI = MFI x percent positive) (E, H, K) for N. gonorrhoeae incubated with the indicated concentrations of 2C7 (C–E), αGc (F–H), and 6B4 (I–K) or highest concentration of each isotype control (iso). Bars indicate the mean ± SD of three independent experiments, with each biological replicate as one data point.

Article Snippet: TABLE 1 Target Antibody/clone name Species, isotype Label a Concentration (mg/ml) Source Use a Comments 2C7 Anti- N. gonorrhoeae LOS IgG3 (-) 0.28 mg/ml S. Ram, UMass IFC, C3 deposition Hybridoma supernatant 6B4 Anti- N. gonorrhoeae LOS IgM, κ (-) 0.33 mg/ml S. Ram, UMass IFC, SBA Hybridoma supernatant CD11b, human M1/70 Rat IgG2b, κ PE 0.2 mg/ml Biolegend Flow cytometry Catalog # 101207 N. gonorrhoeae Rabbit (-) 4-5 mg/ml Meridian IFC, SBA, OPKA Catalog #B65111R IgG, rabbit AffiniPure, Fc-fragment specific Goat AF488 1.5 mg/ml Jackson Immuno Research IFC Catalog # 111-545-008 IgG, mouse Fcy fragment specific Goat AF488 1.5 mg/ml Jackson Immuno Research IFC Catalog # 115-545-164 IgM, mouse μ-Chain specific Goat AF647 1.4 mg/ml Jackson Immuno Research IFC Catalog # 115-606-075 C3, human Goat IgG FITC 7 mg/ml MP Biomedical IFC Catalog # 55167 Isotype control for mouse IgG E1D5H Mouse IgG3 FITC 2.5 mg/ml Cell Signaling Technology IFC Catalog # 37988 Isotype control for mouse IgM Clone MM-30 Mouse IgMκ APC 2-3 mg/ml Biolegend IFC Catalog # 401630 Isotype control for goat anti-human C3 Goat IgG (-) 1 mg/ml R&D Systems IFC Catalog # AB-108-C Open in a separate window a AF, AlexaFluor; APC, allophycocyanin; FITC, fluorescein isothiocyanate; IFC, imaging flow cytometry; OPKA, opsonophagocytic killing; PE, phycoerythrin; SBA, serum bactericidal activity.

Techniques: Binding Assay, Imaging, Flow Cytometry, Incubation, Bacteria, Fluorescence, Concentration Assay, Control

Journal: Infection and Immunity

Article Title: Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae : cross-protective responses elicited by the 4CMenB meningococcal vaccine

doi: 10.1128/iai.00309-23

Figure Lengend Snippet: 4CMenB vaccination of mice elicits cross-reactive antibodies that bind the surface of multiple strains of N. gonorrhoeae. Bacteria of the indicated strain background were incubated with a 1:10 dilution of pooled serum from 4CMenB-vaccinated or alum-treated mice or mouse IgG isotype control. Bacteria were fixed, processed for imaging flow cytometry, and fluorescence index calculated as in Fig. 1. Bars indicate the mean ± SD of three independent experiments, with each biological replicate as one data point. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001, and ****P ≤ 0.0001 by ordinary one-way ANOVA with Tukey’s multiple comparison’s test.

Article Snippet: TABLE 1 Target Antibody/clone name Species, isotype Label a Concentration (mg/ml) Source Use a Comments 2C7 Anti- N. gonorrhoeae LOS IgG3 (-) 0.28 mg/ml S. Ram, UMass IFC, C3 deposition Hybridoma supernatant 6B4 Anti- N. gonorrhoeae LOS IgM, κ (-) 0.33 mg/ml S. Ram, UMass IFC, SBA Hybridoma supernatant CD11b, human M1/70 Rat IgG2b, κ PE 0.2 mg/ml Biolegend Flow cytometry Catalog # 101207 N. gonorrhoeae Rabbit (-) 4-5 mg/ml Meridian IFC, SBA, OPKA Catalog #B65111R IgG, rabbit AffiniPure, Fc-fragment specific Goat AF488 1.5 mg/ml Jackson Immuno Research IFC Catalog # 111-545-008 IgG, mouse Fcy fragment specific Goat AF488 1.5 mg/ml Jackson Immuno Research IFC Catalog # 115-545-164 IgM, mouse μ-Chain specific Goat AF647 1.4 mg/ml Jackson Immuno Research IFC Catalog # 115-606-075 C3, human Goat IgG FITC 7 mg/ml MP Biomedical IFC Catalog # 55167 Isotype control for mouse IgG E1D5H Mouse IgG3 FITC 2.5 mg/ml Cell Signaling Technology IFC Catalog # 37988 Isotype control for mouse IgM Clone MM-30 Mouse IgMκ APC 2-3 mg/ml Biolegend IFC Catalog # 401630 Isotype control for goat anti-human C3 Goat IgG (-) 1 mg/ml R&D Systems IFC Catalog # AB-108-C Open in a separate window a AF, AlexaFluor; APC, allophycocyanin; FITC, fluorescein isothiocyanate; IFC, imaging flow cytometry; OPKA, opsonophagocytic killing; PE, phycoerythrin; SBA, serum bactericidal activity.

Techniques: Bacteria, Incubation, Control, Imaging, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: PD-L1 immunostaining scoring for non-small cell lung cancer based on immunosurveillance parameters

doi: 10.1371/journal.pone.0196464

Figure Lengend Snippet:

Article Snippet: CD68 (PG-M1) , Dako , M0876 , Mouse IgG3, k , 0.06 , 32 min, 37°C , Biolegend, Cat. 401302 , Standard CC1 , OptiView DAB , Hematoxylin II.

Techniques: Concentration Assay, Incubation, Control