Journal: Molecules

Article Title: Recombinant Lactaptin Induces Immunogenic Cell Death and Creates an Antitumor Vaccination Effect in Vivo with Enhancement by an IDO Inhibitor

doi: 10.3390/molecules25122804

Figure Lengend Snippet: Distribution of immune cell populations in peripheral blood. PBMCs were harvested from whole blood samples, stained with CD45, CD3, and CD19 markers, and analyzed by flow cytometry. The number of positive cells is shown as a percentage of total blood CD45+ lymphocytes. ( a ) B-lymphocytes; ( b ) T-lymphocytes. Healthy mice were used as a negative control; “Control vaccination” mice were vaccinated with live MX-7 cells. Statistical differences between control and experimental groups are indicated by * for p < 0.05; ** for p < 0.01.

Article Snippet: The following antibodies and chemicals were obtained from commercial sources: rabbit ant-h/m/rat CRT (Abcam, ab2907, Cambridge, UK), rabbit ant-h/m HMGB1 (Abcam, EPR3507, Cambridge, UK), goat anti-h/m/r GAPDH (R&D Systems, AF5718, Minneapolis, USA), mouse anti-human b-Tubulin (Sigma-Aldrich, T8328, St. Louis, MO, USA), mouse ant-h/rat HSP70 (R&D Systems, 841680, Minneapolis, USA), Alexa Fluor 594-conjugated chicken anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA), Alexa Fluor 555-conjugated goat anti-mouse antibodies (Invitrogen, Carlsbad, CA, USA), polyclonal rabbit-anti-mouse and mouse-anti-rabbit HRP-conjugated antibodies (Biosan, Novosibirsk, Russia), anti-mouse CD45 PerCP/Cy5.5 (Sony Biotechnologies, 1338530, San Jose, CA, USA), anti-mouse CD19 APC (Sony Biotechnologies, 1177650, San Jose, CA, USA), anti-mouse CD3 FITC (Sony Biotechnologies, 1101015, San Jose, CA, USA), anti-mouse CD25 PE (Sony Biotechnologies, 1110040, San Jose, CA, USA), and anti-mouse CD8a PE (R&D Systems, FAB116P, Minneapolis, USA).

Techniques: Staining, Flow Cytometry, Negative Control, Control

Journal: Thoracic Cancer

Article Title: Experimental model of micronodular thymic neoplasm with lymphoid stroma

doi: 10.1111/1759-7714.14716

Figure Lengend Snippet: Lymphoid cells are dependent on epithelial cells in vitro. (a) Floating lymphoid cells were separated from epithelial cells by aspiration and then cultured for 16 and 40 h. The number of cells undergoing apoptosis was quantified with a FITC‐conjugated annexin V and PI assay. Note the populations of cells: viable and not undergoing apoptosis (annexin V‐FITC and PI negative); undergoing apoptosis (annexin V‐FITC positive and PI negative); and end‐stage apoptosis or already dead (annexin V‐FITC and PI positive). (b) Annexin V‐FITC and PI staining of lymphoid cells analyzed by confocal fluorescence microscopy. Blue indicates nuclear staining by DAPI. Scale bar represents 20 μm. (c) DNA fragmentation assay in agarose gel. Note the apoptosis ladder in the lymphoid cells separated from epithelial cells (lane 3) but not in the coculture with epithelial cells (lane 2). 50 bp DNA ladder molecular marker is shown in lane 1.

Article Snippet: Immunofluorescent cytochemical staining was performed as previously described., Rat antimouse CD3 (clone 17A2) conjugated with fluorescein isothiocyanate (FITC) (catalog no. 1101015), rat anti‐mouse CD4 (clone GK1.5) conjugated with phycoerythrin (PE) (catalog no. 1102040), and rat anti‐mouse CD8 (clone 53–6.7) conjugated with FITC (catalog no. 1103525) were purchased from Fujifilm Wako Pure Chemical Co.

Techniques: In Vitro, Cell Culture, Staining, Fluorescence, Microscopy, DNA Fragmentation Assay, Agarose Gel Electrophoresis, Marker

Journal: Vaccines

Article Title: Leptospira Lipid A Is a Potent Adjuvant That Induces Sterilizing Immunity against Leptospirosis

doi: 10.3390/vaccines11121824

Figure Lengend Snippet: PLA is a potent adjuvant that enhances the immune response against Leptospira surface antigen. Mice were immunized with LAV formulated in alum with or without PLA or MPLA, and LAV-specific humoral and cellular immune response was determined as described in Materials and Methods. ( A ) Evaluation of LAV-specific antibody response : the level of LAV-specific antibodies (IgG, IgG1, and IgG2c) in the serum (1:10,000 dilution) obtained on day 28 was analyzed via an ELISA, as detailed in the Materials and Methods Section. ( B ) Assessment of LAV-specific lymphocyte proliferation: On day 28, splenocytes from the different immunized groups were isolated and stimulated with LAV for 72 hrs. Cell proliferation was evaluated using the trypan blue size exclusion assay. ( C ) Analysis of cytokine production: splenocytes were stimulated with LAV (5 µg/mL) for 48 hrs, and the release of IL-4 and IFN-γ cytokines in the culture supernatant was quantified using a sandwich ELISA kit following the manufacturer’s instructions. ( D ) Examination of T cell phenotype: splenocytes stimulated with LAV (2.5 µg/mL) for 24 h were stained with anti-mouse CD3, CD4, and CD8 and then analyzed using flow cytometry. ( E ) CTL assay: The activity of LAV-specific CTLs obtained from various groups was determined using the LDH cytotoxicity kit as specified in the Materials and Methods section. Significant differences were determined using one-way ANOVA. Statistical significance is denoted as “***” for p ≤ 0.001.

Article Snippet: Immunostaining was performed using the following antibodies: Rat anti-mouse FITC-conjugated CD3 (555274, BD Biosciences, 1:300), rat anti-mouse Per-CP-conjugated CD45R (B220; 130-102-815, Miltenyi Biotec, San Jose, CA, USA, 1:100), rat anti-mouse PE-conjugated GC B-Cell marker (GL-7; 561530, BD Biosciences, 1:200), and rabbit anti-mouse Follicular Helper T cell marker CXCR5 (ab254415, Abcam, 1:500).

Techniques: Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, Exclusion Assay, Sandwich ELISA, Staining, Flow Cytometry, CTL Assay, Activity Assay

Journal: Vaccines

Article Title: Leptospira Lipid A Is a Potent Adjuvant That Induces Sterilizing Immunity against Leptospirosis

doi: 10.3390/vaccines11121824

Figure Lengend Snippet: Determination of the long-term immune memory induced by PLA-based vaccine formulation. The animals immunized with PBS, LAV-alum, LAV-alum-MPLA, or LAV-alum-PLA and boosted on the 21st day were bled every week for 24 weeks. The animals were euthanized or given additional antigen boosters (without adjuvant), and spleens were collected after one week to determine memory T cells. ( A ) The humoral (antibody) memory response: the levels of LAV-specific IgG at various time points or isotypes (IgG1 or IgG2c) in the serum were analyzed via an ELISA as described in the Materials and Methods. ( B ) The T cell memory response: splenocytes isolated from mice euthanized at 24 w and 25 w were stimulated with LAV for 72 h; cell proliferation was assessed via trypan blue size exclusion assay. ( C ) Estimation of cytokine levels: 48 hrs after splenocyte stimulation with antigens (LAV; 5 µg/mL), using ELISA, the IL-4 and IFN-γ cytokines in culture supernatant as per the manufacturer’s instructions. ( D ) Analysis of memory phenotype: The splenocytes were stained with fluorescent-labeled antibodies specific against CD3, CD4, CD8, CD62L, and CD44, and we then performed flow cytometry analysis. Representative flow cytometry plots demonstrated the gating strategy for analyzing central (CD44 high and CD62L high ) and effector (CD44 high , CD62L low ) memory response in CD4 and CD8 T cell populations. The numbers of positive cells expressing CD44 and CD62L over the total of CD4+/CD8+ T cells were plotted as histograms. The significant differences were calculated using one-way or two-way ANOVA, with the symbols ****, ***, **, *, and ns indicating p ≤ 0.0001, p ≤ 0.001, p ≤ 0.001, p ≤ 0.05, and non-significant, respectively.

Article Snippet: Immunostaining was performed using the following antibodies: Rat anti-mouse FITC-conjugated CD3 (555274, BD Biosciences, 1:300), rat anti-mouse Per-CP-conjugated CD45R (B220; 130-102-815, Miltenyi Biotec, San Jose, CA, USA, 1:100), rat anti-mouse PE-conjugated GC B-Cell marker (GL-7; 561530, BD Biosciences, 1:200), and rabbit anti-mouse Follicular Helper T cell marker CXCR5 (ab254415, Abcam, 1:500).

Techniques: Formulation, Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, Exclusion Assay, Staining, Labeling, Flow Cytometry, Expressing

Journal: Vaccines

Article Title: Leptospira Lipid A Is a Potent Adjuvant That Induces Sterilizing Immunity against Leptospirosis

doi: 10.3390/vaccines11121824

Figure Lengend Snippet: Formation of Germinal Centers (GCs) in the draining lymph nodes. The formation of GCs in DLNs of various groups was assessed on the 28th day post-immunization. Frozen tissue sections of DLNs were processed and later stained with fluorochrome-conjugated anti-mouse CD3/CD45R/B220/GL-7/CXCR5 antibodies as described in the methodology. The sections were mounted and examined under a fluorescent microscope using tile scanning and then processed using the ZEN software version 3.7 (stitching tool) as described in the methodology section. Data represent three different experiments. Significant differences were calculated using the one-way ANOVA (****, ***, *, and ns indicate p ≤ 0.0001, p ≤ 0.001, p ≤ 0.05, and non-significant, respectively).

Article Snippet: Immunostaining was performed using the following antibodies: Rat anti-mouse FITC-conjugated CD3 (555274, BD Biosciences, 1:300), rat anti-mouse Per-CP-conjugated CD45R (B220; 130-102-815, Miltenyi Biotec, San Jose, CA, USA, 1:100), rat anti-mouse PE-conjugated GC B-Cell marker (GL-7; 561530, BD Biosciences, 1:200), and rabbit anti-mouse Follicular Helper T cell marker CXCR5 (ab254415, Abcam, 1:500).

Techniques: Staining, Microscopy, Software

Journal: Clinical and Developmental Immunology

Article Title: Reevaluating the Concept of Treating Experimental Tumors with a Mixed Bacterial Vaccine: Coley's Toxin

doi: 10.1155/2012/230625

Figure Lengend Snippet: (a)

Article Snippet: Phenotyping of murine immune cells was conducted from blood samples and spleens, following lysis of erythrocytes in lysis buffer (0.17 M Tris, 0.16 M NH 4 Cl) and labeling with the following FITC-conjugated rat antimouse mAbs: CD3, CD11b, CD19 (1 μ g, Immunotools), hamster antimouse mAbs: CD11c (1 μ g, Miltenyi Biotec, Bergisch-Gladbach, Germany) and PE-conjugated rat antimouse mAbs: CD4, CD8, Gr1 (Ly6G) (Miltenyi Biotec), and g/d TCR.

Techniques:

Journal: Clinical and Developmental Immunology

Article Title: Reevaluating the Concept of Treating Experimental Tumors with a Mixed Bacterial Vaccine: Coley's Toxin

doi: 10.1155/2012/230625

Figure Lengend Snippet: (b)

Article Snippet: Phenotyping of murine immune cells was conducted from blood samples and spleens, following lysis of erythrocytes in lysis buffer (0.17 M Tris, 0.16 M NH 4 Cl) and labeling with the following FITC-conjugated rat antimouse mAbs: CD3, CD11b, CD19 (1 μ g, Immunotools), hamster antimouse mAbs: CD11c (1 μ g, Miltenyi Biotec, Bergisch-Gladbach, Germany) and PE-conjugated rat antimouse mAbs: CD4, CD8, Gr1 (Ly6G) (Miltenyi Biotec), and g/d TCR.

Techniques: