Journal: British Journal of Cancer

Article Title: Early immunisation with dendritic cells after allogeneic bone marrow transplantation elicits graft vs tumour reactivity

doi: 10.1038/bjc.2013.39

Figure Lengend Snippet: The impact of immunisation with donor DC on quantitative and qualitative immune reconstitution. Comparative analysis of tumour-bearing mice undergoing allogeneic bone marrow transplantation (allo-BMT) and with immunisation using donor DC on day +7 (BMT+DC), as compared with naïve C57Bl/6 control mice without any experimental manipulation. Spleens were evaluated at 4 weeks post transplant for: ( A ) Reconstitution of spleen cellularity and fractions of CD4 + and CD8 + T cells at 4 weeks after allogeneic BMT ( n =7) and following immunisation with tumour-pulsed donor DC ( n =7), as compared with naïve B6 donors ( n =5). ( B ) Interferon- γ (IFN- γ ) secretion in the corresponding experimental groups ( n =4 in each group). ( C ) Proliferation of splenic lymphocytes in response to mitogenic stimulation with ConA as determined from CFSE dilution and quantified with the ModFit software: naïve B6 mice ( n =5), allografted mice ( n =8) and with DC immunisation ( n =9). ( D ) Neuro-2a cell lysis by effector lymphocytes from spleens of mice in the three experimental groups, and with restimulation in vitro using naïve (antigen-inexperienced, DC naive ) and tumour-pulsed donor DC (DC Neuro2a ). Tumour cell lysis was determined from LDH release by splenic lymphocytes at a target:effector ratio of 1 : 50 representing five mice in each group.

Article Snippet: Interferon- γ was determined in supernatant of lymphocyte suspensions in 96-well microtiter plates using the murine IFN- γ ELISA kit (BD Pharmingen).

Techniques: Transplantation Assay, Software, Lysis, In Vitro

Journal: bioRxiv

Article Title: Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

doi: 10.1101/2022.11.03.515007

Figure Lengend Snippet: Mice were immunized (priming) with chimeric proteins either alone (pO, pNO), or bound to T5 CLP (pO-CLP, pNO-CLP) or supplemented with CFA. Mice were subsequently boosted under the same conditions (except that CFA was replaced by incomplete Freund adjuvant, ICF). (a-b) IFNγ-producing splenocytes were quantified by ELISPOT and ( c ) IFNγ production by splenocytes was measured by ELISA after in vitro restimulation with Ova 257-264 peptide 10 days after boosting. The interval between priming and boosting was of two ( a ) or six months ( b ). The bars correspond to the mean of each group (n = 5-6) and the circles to results of individual mice. ns , non-significant; **, p < 0 . 01 ; ***, p < 0 . 001 .

Article Snippet: For ELISPOT, plates were revealed with supplied reagents (murine IFNγ ELISPOT kit, Diaclone) and spots were counted with the ImmunoSpot® S6 FluoroSpot Line Plate Reader (C.T.L.).

Techniques: Adjuvant, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: BioMed Research International

Article Title: Induction of Antibodies and T Cell Responses by a Recombinant Influenza Virus Carrying an HIV-1 TatΔ 51–59 Protein in Mice

doi: 10.1155/2014/904038

Figure Lengend Snippet: Tat-specific T cell responses in mice vaccinated with WSN/TatΔ 51–59 virus. BALB/c mice ( n = 5/group) were immunized i.n. or i.vag. with WSN/TatΔ 51–59 virus and 7 days later Tat-specific T cell responses were measured in cells from distinct pools of draining lymph nodes, MLN and ILN, respectively, and freshly isolated splenocytes by use of an ex vivo IFN- γ ELISPOT assay with the indicated peptides (a). Splenocytes from mice immunized with WSN/TatΔ 51–59 or WSN virus were cultured for 5 days in the presence of VCF and then washed for the IFN- γ ELISPOT assay. The values reported are those obtained from stimulated cells minus the background from nonstimulated cells. Bars represent the mean values ± SD of triplicate wells. The data are representative of three independent experiments that gave similar results (b). Splenocytes from the above mice were restimulated in the presence of different concentrations of Tat protein for 5 days and the resulting proliferation was assessed by measuring [ 3 H] thymidine incorporation. Results are expressed as the ratio between values (averages of quintuplicates from stimulated and nonstimulated cultures). SI = stimulation index (c).

Article Snippet: Antigen-specific IFN- γ -producing cells were enumerated by using a commercially available murine IFN- γ ELISPOT kit (BD, Pharmingen).

Techniques: Isolation, Ex Vivo, Enzyme-linked Immunospot, Cell Culture

Journal: Cell Reports

Article Title: The C-type Lectin Receptor CLEC12A Recognizes Plasmodial Hemozoin and Contributes to Cerebral Malaria Development

doi: 10.1016/j.celrep.2019.06.015

Figure Lengend Snippet: CLEC12A Recognizes Hemozoin (A and B) ELISA-based binding studies with (A) P. falciparum and (B) P. berghei ANKA. Isolated parasitized RBCs (pRBCs) were lysed, immobilized on microtiter plates, and probed with the indicated fusion proteins. Lysates from uninfected RBCs (uRBCs) and PBS alone (control) were used as controls. Data are presented as mean + SEM and representative of three independent experiments in duplicates for P. falciparum (n = 3) and two experiments in duplicates for P. berghei ANKA (n = 2). (C) Fluorescence microscopy of permeabilized pRBCs. Permeabilized pRBCs were incubated with CLEC12A-hFc, followed by staining with an anti-human immunoglobulin G (IgG) (Fc) Alexa Fluor 488-labeled antibody (AF488, green). Parasitic DNA was stained with DAPI (blue). Hemozoin fluorescence was visualized using a 633-nm HeNe laser (hemozoin, red). pRBCs probed with hFc served as a negative control. Data are representative of two independent experiments (n = 2). Scale bars represent 1 μm. (D) Flow cytometry-based binding studies with hemozoin. Hemozoin was probed with the indicated fusion proteins. CLEC7A-hFc served as a negative control. Means + SD of three independent experiments (n = 3) are shown. (E) Fluorescence microscopy of synthetic hemozoin. Hemozoin was probed with the indicated hFc fusion proteins and incubated with an anti-human IgG (Fc) Alexa Fluor 488 antibody (AF488, green). Samples incubated with hFc served as a negative control. Data are representative of two independent experiments (n = 2). Scale bars represent 10 μm. (F) Recognition of hemozoin (100 μg/mL) and MSU (250 μg/mL) by human (h) and murine (m) CLEC12A-CD3 reporter cell lines. A murine CLEC7A-CD3 reporter cell line served as a negative control. Data are presented as mean + SD and are representative of three independent experiments (n = 3) in triplicates (two experiments) or quadruplicates (one experiment). Statistical significance was evaluated using unpaired (ELISA, reporter cell assay) or paired (flow cytometry-based binding studies) two-tailed Student’s t test. Asterisks indicate significant differences ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). See also Figure S1 .

Article Snippet: Murine IFN-γ Standard ABTS ELISA Development Kit , PeproTech , CAT#900-K98.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Isolation, Control, Fluorescence, Microscopy, Incubation, Staining, Labeling, Negative Control, Flow Cytometry, Two Tailed Test

Journal: Cell Reports

Article Title: The C-type Lectin Receptor CLEC12A Recognizes Plasmodial Hemozoin and Contributes to Cerebral Malaria Development

doi: 10.1016/j.celrep.2019.06.015

Figure Lengend Snippet:

Article Snippet: Murine IFN-γ Standard ABTS ELISA Development Kit , PeproTech , CAT#900-K98.

Techniques: Recombinant, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Cell Isolation, Software

Journal: Biomacromolecules

Article Title: Cytosolic Delivery Mediated via Electrostatic Surface Binding of Protein, Virus, or siRNA Cargos to pH-Responsive Core−Shell Gel Particles

doi: 10.1021/bm801199z

Figure Lengend Snippet: Figure 3. Cytosolic delivery of ova for OT-I T cell priming. (A) BMDCs were incubated with different concentrations of soluble ova (O), ova- coated PDEAEMA CSPs (2), or ova-coated PMMA CSPs (9) in complete medium containing 10% FBS, before being washed and mixed with naı¨ve OT-1 ova-specific CD8+ T-cells. The concentration of particles was fixed at 25 µg/mL for all ova doses. IFN-γ secreted by the T-cells in response to antigen presentation by the DCs was measured by ELISA after 3 days. Shown are mean ( SD of triplicate samples. (B) BMDCs were pulsed with ova in different forms: soluble ova (10 µg/mL) for 1 h or during an entire 3 day coculture, ova mixed with PDEAEMA or PMMA CSPs (without prebinding to particles, ova + CSPs), or ova adsorbed to PDEAEMA or PMMA CSPs prior to addition to DCs (ova-CSPs) in complete medium containing 10% FBS. DCs were washed following antigen pulsing and then cocultured with OT-I T-cells. IFN-γ secreted by T-cells in response to antigen presentation by the DCs was measured by ELISA after 3 days. Shown are mean ( SD of triplicate samples.

Article Snippet: Interferon gamma (IFN-γ) in the supernatants of these cultures was then quantified using an ELISA kit (murine Interferon gamma (IFNγ) Duoset ELISA Development Kit, R&D Systems) according to the manufacturer’s instructions.

Techniques: Incubation, Concentration Assay, Immunopeptidomics, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Design of SARS-CoV-2 RBD mRNA Vaccine Using Novel Ionizable Lipids

doi: 10.1101/2020.10.15.341537

Figure Lengend Snippet: Female BALB/c mice were immunized either i.m. or i.d. with 5μg LNPs-RBD mRNA or s.c. with 10μg rRBD, and boosted with an equivalent dose 25 days later. Serum and spleen were collected at days 23 (“pre-boost”) and 39 (“post-boost”) after initial vaccination. Panel A-Schematic diagram of immunization and sample collection. Panel B-SARS-CoV-2 spike-specific IgG antibody titer was determined by ELISA. Panel C-PRNT 50 titers were determined post-boost using a VSV-based pseudovirus PRNT assay. Panel D-SARS-CoV-2 spike-specific cellular response was determined by ELISpot. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparisons test (for ELISA data) or an unpaired two-tailed Student’s t-test (for PRNT 50 and ELISpot data) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Cells were incubated with antigens for 24 h, and the frequency of IFNγ-secreting cells was determined using Murine IFNγ Single-Color Enzymatic ELISPOT kit (CTL, #MIFNG 1M/5) with strict adherence to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Plaque Reduction Neutralization Test, Enzyme-linked Immunospot, Two Tailed Test