Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Structural organization of canine Peyer’s patches, according to immunofluorescence data. ( A ) Sample collection and preparation. Intestinal tissues from two dogs were sampled, and regions of ∼2 cm in length were identified as Peyer’s patches (PPs). The PPs were isolated, sectioned on a cryostat, and prepared for microscopy, with the various structures within the intestine identified before DAPI staining. ( B ) Visualization of PPs with H&E show different staining intensities within the follicles. The staining with Ki67, for proliferating cells, and CD21, for B cells, allows the visualization of proliferating B cells within the GC. ( C ) Immunofluorescence staining of PPs. PPs were stained with DAPI in combination with Abs against CD8 and one of the following markers: CD21, CD4, or FOXP3. Epifluorescence microscopy is shown at original magnification ×10, visualizing specific structures, including the subepithelial dome (SED), T cell areas, and B cell follicles. ( D ) Myeloid cell staining. Immunostaining of the CD14 and CD11c markers, along with CD21, was used to identify dendritic cells within the SED. Different images from the lower and upper parts of the sample were captured to adequately show the CD11c staining in proximity to both the epithelium and the follicle.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Immunofluorescence, Isolation, Microscopy, Staining, Epifluorescence Microscopy, Immunostaining

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Characterization of T and B cell populations in canine Peyer’s patches by flow cytometry. ( A ) Peyer’s patches (PPs) from two dogs were dissociated, and single-cell suspensions were stained for flow cytometry in a Cytek Northern Lights flow cytometer. ( B ) Proportions of T and B cells. T cells accounted for 41.9% and B cells for 22.2% of the cell population analyzed. Within the B cell population, 11.6% of the cells were positive for Bcl-6. T cells were further separated into CD4 + , CD8 + , and CD4 + CD8 + subsets, revealing high levels of heterogeneity in the expression of the regulatory marker FOXP3. ( C ) FOXP3 expression in T cell subsets. The percentage of FOXP3 + cells was significantly higher among CD3 + CD4 + CD8 + cells than among CD3 + CD4 + or CD3 + CD8 + cells. Statistical analysis was performed by two-way ANOVA. * p ≤ 0.05, **** p ≤ 0.001. dp, double-positive.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Flow Cytometry, Staining, Northern Blot, Expressing, Marker

Journal: ImmunoHorizons

Article Title: Characterization of Canine Peyer’s Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing

doi: 10.4049/immunohorizons.2300091

Figure Lengend Snippet: Gene expression distribution, B and T cell subpopulations, and the Peyer’s patch atlas. ( A ) Marker expression. The expression of markers such as ICOS, TOP2A, JCHAIN, CCR10, and RORC was visualized across the clusters, facilitating cell subtype identification. ( B ) T cell subpopulations. The distribution of different T cell subpopulations is presented as a pie chart, with the corresponding percentages. ( C ) B cell subpopulations. The distribution of different B cell subpopulations is shown as a pie chart, with the corresponding percentages. ( D ) We propose an annotation of the immune cells identified within clusters, based on the specific expression patterns of marker genes. Bcl-6, B cell lymphoma 6; CD62L, CD62 L-selectin; DZ, dark zone; GC, germinal center; ILC3, innate lymphoid cells group 3; LZ, light zone; PD-1, programmed cell death protein 1; Tfh, T follicular helper cell.

Article Snippet: We used the following Abs: mouse anti-dog CD3 Alexa Fluor 700 (Bio-Rad, MCA1774A700), rat anti-dog CD4 PE-Cy7 (Bio-Rad, MCA1038PeCy7), rat anti-dog CD8 Pacific Blue (Bio-Rad, MCA1039PB), and mouse anti-dog CD21 Alexa Fluor 647 (Bio-Rad, MCA1781A647).

Techniques: Gene Expression, Marker, Expressing

Journal: Vaccine

Article Title: Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection.

doi: 10.1016/j.vaccine.2009.08.091

Figure Lengend Snippet: Fig. 3. Lymphocyte proliferation against CLA. Cells isolated from peripheral blood (PBMC), lymph node and spleen were labeled with CFSE and incubated in presence of CLA, Con A or without exogenous stimuli for 5 days, stained with anti-canine CD4+: RPE or anti-canine CD8+: RPE and proliferative cells were quantified by flow cytometry. Lines over the bars represent the percentage of proliferation in the presence of Con A. Bars represent mean value of the percentage of proliferation in the presence of CLA ± S.E.M. G1: negative control; G2: positive control; G3: pORT-LACK/MVA-LACK vaccinated dogs.

Article Snippet: G1: negative control; G2: positive control; G3: vaccinated dogs (pORT-LACK/MVA-LACK). and stained with Rat anti-canine CD4+: RPE (Serotec) or Rat anticanine CD8+: RPE (Serotec).

Techniques: Isolation, Labeling, Incubation, Staining, Cytometry, Negative Control, Positive Control

Journal: Vaccine

Article Title: Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection.

doi: 10.1016/j.vaccine.2009.08.091

Figure Lengend Snippet: Fig. 4. Lymphocyte proliferation against LACK. Cells isolated from peripheral blood (PBMC), lymph node and spleen were labeled with CFSE and incubated in presence of LACK, Con A or without exogenous stimuli for 5 days, stained with anti-canine CD4+: RPE or anti-canine CD8+: RPE and proliferative cells were quantified by flow cytometry. Lines over the bars represent the percentage of proliferation in the presence of Con A. Bars represent mean value of the percentage of proliferation in the presence of LACK ± S.E.M. G1: negative control; G2: positive control; G3: pORT-LACK/MVA-LACK vaccinated dogs.

Article Snippet: G1: negative control; G2: positive control; G3: vaccinated dogs (pORT-LACK/MVA-LACK). and stained with Rat anti-canine CD4+: RPE (Serotec) or Rat anticanine CD8+: RPE (Serotec).

Techniques: Isolation, Labeling, Incubation, Staining, Cytometry, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

doi: 10.1371/journal.pone.0183398

Figure Lengend Snippet: Mucosal tissues from animal species were processed for immunohistology analysis, as described in Methods. Slides were stained with the following specific antibodies: anti-MHC-II, anti-CD163, anti-CD172a, anti-CD3, and anti-CD4 to detect and quantify positive cells in tissue sections or with toluidine blue to quantify mast cells (magnification x200). Representative photomicrographs of mucosal tissue sections are shown.

Article Snippet: The following polyclonal or monoclonal antibodies (mAbs) were used for immunohistology: anti-CD3 (for rats: clone 1F4, Bio-Rad, Oxford, UK; for dogs: Ab828, Abcam, Cambridge, UK; for minipigs: clone 8E6, WSU Monoclonal antibody center, Pullman, WA; for monkeys: clone CD3-12, Abcam), anti-CD4 (for rats: clone OX-35, Bio-Rad; for dogs: clone DH-29A, WSU Monoclonal antibody center; for minipigs: clone 74-12-4, WSU Monoclonal antibody center; for monkeys: clone BC/1F6, Abcam), anti-CD163 (for all species: clone AM-3K, Antibodies online, Paris, France), anti-CD172a (for rats: clone ED9, Bio-Rad; for dogs: clone DG-DH59B, WSU Monoclonal antibody center; for minipigs: clone BL1H7, Bio-Rad; for monkeys: Ab139698, Abcam), anti-MHC-II (for rats: clone OX-6, Bio-Rad; for dogs: clone DG-H42A, WSU Monoclonal antibody center; for minipigs: clone TH21A, WSU Monoclonal antibody center; for monkeys: clone L243, Abcam).

Techniques: Staining

Journal: PLoS ONE

Article Title: Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

doi: 10.1371/journal.pone.0183398

Figure Lengend Snippet: Mucosal tissues from animal species were processed for immunohistology analysis, as described in Methods. Cell counting was performed on slides labeled with Abs specific for APC (anti-MHC-II, anti-CD163, anti-CD172a) and T cell (anti-CD3 and anti-CD4) markers or stained with toluidine blue for mast cells to evaluate the mean number of positive cells per field using a light microscope (magnification x400). All areas (epithelium (Epith.), Lamina propria (LP) and muscle) were scored. Histograms represent the mean + SEM with n = 3.

Article Snippet: The following polyclonal or monoclonal antibodies (mAbs) were used for immunohistology: anti-CD3 (for rats: clone 1F4, Bio-Rad, Oxford, UK; for dogs: Ab828, Abcam, Cambridge, UK; for minipigs: clone 8E6, WSU Monoclonal antibody center, Pullman, WA; for monkeys: clone CD3-12, Abcam), anti-CD4 (for rats: clone OX-35, Bio-Rad; for dogs: clone DH-29A, WSU Monoclonal antibody center; for minipigs: clone 74-12-4, WSU Monoclonal antibody center; for monkeys: clone BC/1F6, Abcam), anti-CD163 (for all species: clone AM-3K, Antibodies online, Paris, France), anti-CD172a (for rats: clone ED9, Bio-Rad; for dogs: clone DG-DH59B, WSU Monoclonal antibody center; for minipigs: clone BL1H7, Bio-Rad; for monkeys: Ab139698, Abcam), anti-MHC-II (for rats: clone OX-6, Bio-Rad; for dogs: clone DG-H42A, WSU Monoclonal antibody center; for minipigs: clone TH21A, WSU Monoclonal antibody center; for monkeys: clone L243, Abcam).

Techniques: Cell Counting, Labeling, Staining, Light Microscopy

Journal: Toxicology Letters

Article Title: Capture ELISA and flow cytometry methods for toxicologic assessment following immunization and cyclophosphamide challenges in beagles

doi: 10.1016/S0378-4274(00)00173-9

Figure Lengend Snippet: Mean relative% lymphocyte subpopulations at pre-treatment (days −4 and −5) and following CY treatment (days 17, 20 and 22) as determined by flow cytometry: (A) males and (B) females. Relative% B cells were decreased in males on day 22 compared with day −5 ( P <0.05). Relative% T-helper cells (males only) were increased ( P <0.05) compared with day −5 according to a two-sample t -test on days 17, 20 and 22; however, absolute numbers were dramatically decreased.

Article Snippet: The reagents used were: phycoerythrin (PE) anti-human CD21, fluorescein isothiocyanate (FITC) rat IgG 1 , FITC rat IgG 2a ; PE rat IgG 1 (PharMingen, San Diego, CA), and rat anti-canine CD4:FITC, rat anti-canine CD8:PE; rat anti-canine CD5:FITC (Serotec, Raleigh, NC), Dulbecco’s phosphate-buffered saline (Pierce, Rockford, IL), and fetal calf serum (Sigma).

Techniques: Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine

doi: 10.1007/s00262-010-0921-2

Figure Lengend Snippet: Cytometry and cell sorting of primary canine DC populations from PBMCs. Canine PBMCs (60 ml fresh EDTA-treated blood) were labeled for CD4, CD8, and CD11c, analyzed by flow cytometry and sorted into CD11c+/CD4−/CD8+ (b lower right gate), CD11c+/CD4+/CD8− (a lower right gate), CD11c+/CD4−/CD8− (b lower left gate excluding CD11c+/CD4+/CD8− cells in the a lower right gate), and CD11c++/CD4+/CD8+ (b upper left gate) populations enriched in peripheral blood-derived DCs. DC-enriched/labeled populations are indicated by color back-gates. Sorted populations were gated first on whole cells in forward/side scatter dot plots and then analyzed for fluorescence. Typical sorts involved labeling approximately 1–3 × 109 PBMCs recovering 1–5 × 106 CD11c+ cells sufficient to prepare a single hybrid-DC fusion vaccine

Article Snippet: Cells were centrifuged once and resuspended in 4 ml flow wash buffer (FWB: HBS, 10% fetal bovine serum) and incubated at room temperature for 40 min. PBMC populations (1–3 × 10 9 cells in 1 ml) were labeled with antibodies against canine CD4 (FITC-conjugated polyclonal rat anti-canine, Serotec), CD8 (RPE-conjugated polyclonal rat anti-canine, Serotec), and CD11c (monoclonal mouse anti-canine, Serotec) as previously described [ 21 ] in combinations of up to three antibodies (100 μl of each antibody solution per 1 ml reaction volume).

Techniques: Cytometry, FACS, Labeling, Flow Cytometry, Derivative Assay, Fluorescence