Journal: Veterinary pathology

Article Title: Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma.

doi: 10.1177/03009858211042588

Figure Lengend Snippet: Figure 5. Flow cytometric analysis of equine bronchoalveolar lavage fluid (BALF) leukocytes. (a) Indicates that cells from p1 have higher forward scatter (FSC) and side scatter (SSC) than cells from p2, indicating they are larger and more internally complex. (b) Unstained control sample showing that many (red circle) but not all cells from p1 (upper panel) have autofluorescence in the phycoerythrin (PE) and fluorescein isothiocyanate (FITC) channels. However, cells from p2 (lower panel) are not autofluorescent. (c) Using anti-CD90 (neutrophil marker) and anti-CD206 (macrophage marker) antibodies, cells from p1 (upper panel) but not p2 (lower panel) are identified as neutrophils (Q3) and macrophages (Q1), respectively. (d) Using antibodies against CD5 (lymphocyte marker) and PanB cells, cells in p1 (upper panel) are double- negative, and cells in p2 (lower panel) are identified as lymphocytes; the red circle indicates autofluorescent cells.

Article Snippet: The fluorescent primary antibodies used in this study were phycoerythrin (PE) conjugated mouse antihuman CD206 (clone 3.29B1, Beckman Coulter), fluorescein isothiocyanate (FITC) conjugated mouse anti-horse CD5 (clone CVS5, Bio-Rad), and PE conjugated mouse anti-horse PanB cells (clone CVS36, Bio-Rad).

Techniques: Control, Marker

Journal: International Immunology

Article Title: B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

doi: 10.1093/intimm/dxu001

Figure Lengend Snippet: Clinical profile of patients examined in this study

Article Snippet: For CD5 antibody-binding experiments, mouse anti-human CD5 mAb (clone MF7-14–5, azide free) and an appropriate control mAb (clone W3/25, azide free) were used (Serotec, Raleigh, NC, USA).

Techniques: Expressing

Journal: International Immunology

Article Title: B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

doi: 10.1093/intimm/dxu001

Figure Lengend Snippet: CpG and IL-21 induce apoptosis in CD5+ B-CLL cells, but not in CD5− B cells. (A) PBMC from five subjects with CD5+ B-CLL (left panel), two subjects with CD5− B-CLL (middle panel) and four healthy subjects (right panel) were isolated and cultured for 4 days in the presence of CpG, IL-21 or both. Cells were harvested, stained with antibodies to CD19, CD5, annexin V and propidium iodide (PI) and analyzed by flow cytometry. Shown are mean percentages of viable B cells determined by annexin V/PI staining. Error bars indicate SEM for left and right panels and standard deviation for middle panel. (B and C) B-CLL cells from three different donors with high cellularity (>80% CD19+CD5+ B-CLL cells) were incubated with IL-21, CpG or a combination of both, in the presence of increasing concentrations of a monoclonal anti-CD5 antibody. Viability of B-CLL cells was determined by FACS, using annexin V/PI. Line graphs (B) and bar graphs (C) show absolute numbers of viable B-CLL cells on day 4. Error bars indicate SEM from three independent experiments.

Article Snippet: For CD5 antibody-binding experiments, mouse anti-human CD5 mAb (clone MF7-14–5, azide free) and an appropriate control mAb (clone W3/25, azide free) were used (Serotec, Raleigh, NC, USA).

Techniques: Isolation, Cell Culture, Staining, Flow Cytometry, Standard Deviation, Incubation

Journal: International Immunology

Article Title: B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21

doi: 10.1093/intimm/dxu001

Figure Lengend Snippet: GrB production in B cells requires activation of members of the JAK/STAT pathway. (A) Purified B cells from healthy donors or B-CLL patients (>99.5%) were cultured for 15min with IL-21, anti-BCR or CpG. Analysis of pJAK1, pJAK3, pSTAT1 and pSTAT3 was performed by western immunoblotting, each membrane was controlled for correct loading by stripping and restaining the membrane for β-actin (n = 3). (B) Purified healthy B cells and B-CLL cells were cultured for 20min with IL-21 and anti-BCR in the presence or absence of the JAK inhibitor P6. Analysis of pSTAT1 and pSTAT3 was performed by western immunoblotting (n = 3). (C and D) Purified normal B cells and B-CLL cells were cultured for 16h with IL-21 and anti-BCR in the presence or absence of P6. After incubation, cells were stained for intracellular GrB. Shown is GrB production of CD5+CD19+ B-CLL cells and CD19+ B cells as determined by flow cytometry. Panels in (C) illustrate representative dot plots out of five donors (healthy B cells) or four patients (B-CLL). Panels in (D) indicate percentages of GrB+ B cells with each line indicating an individual donor.

Article Snippet: For CD5 antibody-binding experiments, mouse anti-human CD5 mAb (clone MF7-14–5, azide free) and an appropriate control mAb (clone W3/25, azide free) were used (Serotec, Raleigh, NC, USA).

Techniques: Activation Assay, Purification, Cell Culture, Western Blot, Membrane, Stripping Membranes, Incubation, Staining, Flow Cytometry