Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 2: Porcine antigen presenting cells (pAPCs) stimulate proliferation of allogeneic helper and cytotoxic T cells: Irradiated porcine APCs generated using pGM-CSF alone (pGM-CSF APCs) or pGM-CSF combined with pIL- 4 (pGM-CSF/pIL-4 APCs) were cocultured at varying ratios with CFSE-labeled, erythrocyte-depleted allogeneic porcine splenocytes. Cell division for CD4+ve and CD8+ve cells was detected following 96h of culture by three- color flow cytometric analysis using PI exclusion to gate on viable cells, surface staining with mouse antiporcine CD4 or CD8 followed by goat antimouse-PE, and CFSE fluorescence dilution. Examples of flow cytometric plots (following PI gating) for stimulated and unstimulated splenocytes are shown for the CD4+ve

Article Snippet: The following antibody preparations were used: Antimouse CD40 (clone HM403), anti-SLA-DR (clone 1030 hours-1–19), antipig CD4 (clone 74-12-4), antipig CD8 (clone 76-2-11), antihuman CD19-PE (clone HIB19), antimouse CD19-PE (clone 1D3), antimouse CD8-PE (clone 53–6.7), antimouse CD4FITC (clone GK1.5), biotinylated anti mouse CD90 (Thy1.2, clone 53-2-1), streptavidin-PE, antimouse IL-10R (clone 1B1), and RatIgG1 (clone R3-34); all purchased from BD PharMingen International, San Diego, CA; goat antimouse IgG-FITC (Southern Biotechnology Associates, Inc. Birmigham, AL); goat F (ab0)2 antimouse Ig (Tago, Inc. Burlingame, CA); affinity purified goat antipig IgG (Bethyl Laboratories Inc., Montgomery, TX); biotinylated goat antipig IgG (Vector Laboratories, Burlingame, CA).

Techniques: Irradiation, Generated, Labeling, Staining, Fluorescence

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 8: Porcine antigen presenting cells (pAPCs)-induced murine B-cell activation and proliferation is not mediated by porcine IL-12, IL-6, or IL-10. (A) Purified CFSE-labeled murine B cells were incubated with control medium (A) or with 25% medium from pGM-CSF APCs (pAPC medium) containing various antibodies: rabbit IgG (B), rabbit antipIL-12 (C), rabbit antipIL-6 (D), rat IgG1 (E), rat antimouse IL10R (F), rabbit IgG and rat IgG1 (G), and all three anticytokine antibodies (H). After 72 h viability (upper graph), B-cell enlargement (Forward scatter: middle graph), and proliferation (% divided: lower graph) were analyzed. Results are expressed as mean ± SD of triplicate samples for each condition. Addition of pAPC medium resulted in significant increases in B-cell viability, Forward Scatter, and B-cell division compared with control cultures. The addition of cytokine-blocking antibodies separately or in combination was not associated with significant reductions in any of the three indices when compared with the effects of appropriate control antibodies. (B) A similar experiment was carried out with the additional presence of anti-CD40 2.5 mg/mL in all conditions. B-cell division was measured on the basis of CFSE dilution and expressed as mean percent divided ± SD of triplicate samples for each condition. Addition of pAPC was associated with significantly increased B-cell division compared with control conditions. Neither control antibodies nor cytokine- blocking antibodies were associated with reductions in the degree to which pAPC medium enhanced CD40-induced B-cell proliferation. *Values for pAPC medium-treated cultures that are significantly greater (p < 0.05) than corresponding results for untreated cultures.

Article Snippet: The following antibody preparations were used: Antimouse CD40 (clone HM403), anti-SLA-DR (clone 1030 hours-1–19), antipig CD4 (clone 74-12-4), antipig CD8 (clone 76-2-11), antihuman CD19-PE (clone HIB19), antimouse CD19-PE (clone 1D3), antimouse CD8-PE (clone 53–6.7), antimouse CD4FITC (clone GK1.5), biotinylated anti mouse CD90 (Thy1.2, clone 53-2-1), streptavidin-PE, antimouse IL-10R (clone 1B1), and RatIgG1 (clone R3-34); all purchased from BD PharMingen International, San Diego, CA; goat antimouse IgG-FITC (Southern Biotechnology Associates, Inc. Birmigham, AL); goat F (ab0)2 antimouse Ig (Tago, Inc. Burlingame, CA); affinity purified goat antipig IgG (Bethyl Laboratories Inc., Montgomery, TX); biotinylated goat antipig IgG (Vector Laboratories, Burlingame, CA).

Techniques: Activation Assay, Purification, Labeling, Incubation, Control, Blocking Assay

Journal: Veterinární Medicína

Article Title: Humoral and cellular immune response to Histophilus somni recombinant heat shock protein 60 kDa in farm animals

doi: 10.17221/8528-vetmed

Figure Lengend Snippet: Figure 1. SDS-PAGE and immunoblotting of rHsp60 kDa H. somni protein with 6xHistag MoAb (1 : 2000) and goat anti-mouse IgG (1 : 2000) HRPO conjugate. A = marker page ruler prestained protein ladder (5 µl), B = purified rHsp60 protein (20 µl), 1 = SDS-PAGE, 2 = immunoblot ing with 6xHistag monoclonal antibody

Article Snippet: Plates were incubated at room temperature for 2 h. Goat anti-chicken IgG (Sigma Aldrich, cat. no. A-9046) diluted 1 : 20 000, antipig IgG (1 : 50 000), IgM (1 : 25 000), IgA (1 : 25 000) (Bethyl Laboratories, cat. no. E100-104, E100-100, E100-102, respectively) and sheep anti-bovine IgG1 (1 : 40 000), IgG2 (1 : 20 000), IgM (1 : 20 000, AbD Serotec, cat. no. AAI21P, AAI22P, AAI19P, respectively) HRPO conjugates in PBS-T were used.

Techniques: SDS Page, Western Blot, Marker, Purification