Journal: Biomedicines

Article Title: Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19

doi: 10.3390/biomedicines10030630

Figure Lengend Snippet: List of fluorochrome-labeled antibodies used in flow cytometry studies. FITC, fluorescein isothiocyanate; PE, phycoerythrin; PE/Cy7, tandem comprising phycoerythrin and cyanine 7; APC, allophycocyanin; APC/Cy7, tandem comprising allophycocyanin and cyanine 7.

Article Snippet: INF-γ , 45-15 , PE , Miltenyi Biotec , 130-113-493.

Techniques: Flow Cytometry

Journal: Analytical Chemistry

Article Title: Detecting Cytokine Release from Single T-cells

doi: 10.1021/ac901390j

Figure Lengend Snippet: Figure 1. (A) Fabrication of hydrogel microwells for capturing single T-cells and secreted cytokines. Step 1. Anti-CD4 and anti-IFN-γ Abs are mixed in solution and physiadsorbed onto a lyophilized, PEG hydrogel-coated glass slide. Step 2. Photosensitive PEG prepolymer is spin- coated on top of the Ab-containing hydrogel layer and then exposed to UV through a photomask. Step 3. Prepolymer not exposed to UV is removed by development in water, leaving behind microwells with nonfouling walls and Ab-containing attachment sites. (B) Detection of IFN-γ secreted by single T-cells. Step 1. PEG hydrogel microwells are enclosed inside a PDMS microfluidic device and are incubated with RBC- depleted human blood. Step 2. Controlled washing is used to remove nonspecific cells, leaving behind Ab-bound T-cells. Step 3. Mitogenic activation induces cytokine production in T-cells. Secreted cytokines become captured in the proximity of cells and are detected using sandwich immunoassay.

Article Snippet: Monoclonal antibodies used for capturing Tlymphocytes and cytokines consisted of the following: purified mouse antihuman CD4 Abs (13B8.2) from Beckman-Coulter (Fullerton, CA), and purified mouse antihuman IFNγ Ab (clone K3.53), biotinylated goat antihuman IFN-γ Ab from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Activation Assay

Journal: Analytical Chemistry

Article Title: Detecting Cytokine Release from Single T-cells

doi: 10.1021/ac901390j

Figure Lengend Snippet: Figure 2. Comparing sensitivity of IFN-γ immunoassay. Ab spots comprised of anti-IFN-γ or anti-CD4 and -IFN-γ were printed into PEG hydrogel-covered glass slides and incubated with varying concentra- tions of human recombinant IFN-γ. Coimmobilizing two Ab types did not affect characteristics of IFN-γ immunoassay.

Article Snippet: Monoclonal antibodies used for capturing Tlymphocytes and cytokines consisted of the following: purified mouse antihuman CD4 Abs (13B8.2) from Beckman-Coulter (Fullerton, CA), and purified mouse antihuman IFNγ Ab (clone K3.53), biotinylated goat antihuman IFN-γ Ab from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Recombinant

Journal: Analytical Chemistry

Article Title: Detecting Cytokine Release from Single T-cells

doi: 10.1021/ac901390j

Figure Lengend Snippet: Figure 3. Capturing T-cells and detecting IFN-γ release on Ab-containing surfaces. Solution containing a mixture of anti-CD4 and -IFN-γ Abs was pipetted onto a PEG hydrogel-covered glass slide creating ∼1 mm diameter Ab spots. An Ab-modified surface was placed inside a microfluidic device, incubated with RBC-depleted whole blood and then mitogenically activated. (A) Immunofluorescent staining with anti-CD3-FITC - reveals that majority of captured cells stain positive (green) for this T-cell marker. T-cells are also stained with DAPI (blue) to reveal morphology of the nucleus. (B-C) Immunofluorescent staining with anti-IFN-γ-biotin and streptavidin-Alexa546 shows detection of secreted IFN-γ in the vicinity of surface bound T-cells (red fluorescence). Comparison of mitogenic activation lasting 4 h (B) and 15 h (C) shows a stronger cytokine signal (red) corresponding to longer activation.

Article Snippet: Monoclonal antibodies used for capturing Tlymphocytes and cytokines consisted of the following: purified mouse antihuman CD4 Abs (13B8.2) from Beckman-Coulter (Fullerton, CA), and purified mouse antihuman IFNγ Ab (clone K3.53), biotinylated goat antihuman IFN-γ Ab from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Staining, Marker, Comparison, Activation Assay

Journal: Analytical Chemistry

Article Title: Detecting Cytokine Release from Single T-cells

doi: 10.1021/ac901390j

Figure Lengend Snippet: Figure 4. PEG hydrogel microwells containing T-cell capture (anti- CD4) and cytokine detection (anti-IFN-γ) Abs. (A) Micropatterned surfaces were stained with FITC-labeled antimouse IgG to show localization of the Abs (green fluorescence) at the bottom of PEG hydrogel microwells. (B) To reveal presence of cytokine-sensing Abs, micropatterned surfaces were challenged with human recombinant IFN- γ (500 ng/mL) and then incubated with anti-IFN-γ-biotin and streptavidin-Alexa546 (red fluorescence). (C) Hydrogel microwells were enclosed in a microfluidic device and incubated with RBC- depleted human blood, resulting in capture of the cells. This image highlights the possibility to sequester single cells by defining the area of an attachment site. Majority of cells stained positive for CD3 marker (green fluorescence) pointing to T-lymphocyte phenotype. (D) SEM image showing an array of single T-cells residing in 20 µm diameter hydrogel microwells.

Article Snippet: Monoclonal antibodies used for capturing Tlymphocytes and cytokines consisted of the following: purified mouse antihuman CD4 Abs (13B8.2) from Beckman-Coulter (Fullerton, CA), and purified mouse antihuman IFNγ Ab (clone K3.53), biotinylated goat antihuman IFN-γ Ab from R&D Systems (Minneapolis, MN).

Techniques: Staining, Labeling, Recombinant, Incubation, Marker

Journal: Analytical Chemistry

Article Title: Detecting Cytokine Release from Single T-cells

doi: 10.1021/ac901390j

Figure Lengend Snippet: Figure 5. Detection of IFN-γ production from single T-cells. Cells were captured on micropatterned surfaces inside a microfluidic device and then activated with mitogens for 15 h. (A) Brightfield image of single T-cells captured from peripheral blood inside the PEG hydrogel microwells. (B) Staining with anti-CD4-PE (red) shows that all of the captured cells are CD4 positive T-cells. (C) The same image stained with anti-IFN- γ-biotin and neutravidin-FITC shows “halo” of green fluorescence due to secreted cytokine. The IFN-γ concentration in the vicinity of single T-cells was estimated to be 661 ( 75 ng/mL (39 ( 4.4 nM).

Article Snippet: Monoclonal antibodies used for capturing Tlymphocytes and cytokines consisted of the following: purified mouse antihuman CD4 Abs (13B8.2) from Beckman-Coulter (Fullerton, CA), and purified mouse antihuman IFNγ Ab (clone K3.53), biotinylated goat antihuman IFN-γ Ab from R&D Systems (Minneapolis, MN).

Techniques: Staining, Concentration Assay

Journal: The veterinary quarterly

Article Title: Holstein × Montbéliarde-sired F1 generation crossbred female calves have an increased cellular immune response potential compared with purebred Holsteins.

doi: 10.1080/01652176.2024.2435982

Figure Lengend Snippet: Figure 2. Gating strategies to define bovine T cell subsets and their IFN-γ expression. Mononuclear cells were isolated from blood of 3 months-old female calf and surface stained with antibody cocktails (CD4, CD8, CD44, IFN-γ) after PMA/ionomycin mixture stimulation. The lymphocytes are chosen with FSC versus SSC (A) and then singlets are gated using FSC-a and FSC-H (B). By outputting CD4 versus CD8, CD4+ CD8-, CD8+ CD4- are identified, respectively (C). The CD4+ CD8- T cells expressing IFN-γ (D). The CD8+ CD4- T cells expressing IFN-γ (G). CD4+ CD8- T cells and CD4+ CD8- T cells can be further subdivided into CD44+ and CD44- subsets (E and F), respectively. The lymphocytes expressing IFN-γ (H).

Article Snippet: Monoclonal antibodies (mAb) specific for bovine TCRγδ (WC1), CD8, CD4 and IFN-γ with different fluorochrome conjugates were purchased from BIO-RAD (BIO-RAD, Shanghai, China) and mAb to human CD27, mouse CD44 were purchased from Biolegend (Biolegend, San Diego, USA) (Table 1).

Techniques: Expressing, Isolation, Staining

Journal: The veterinary quarterly

Article Title: Holstein × Montbéliarde-sired F1 generation crossbred female calves have an increased cellular immune response potential compared with purebred Holsteins.

doi: 10.1080/01652176.2024.2435982

Figure Lengend Snippet: Figure 5. Differences of IFN-γ expression in lymphocytes and their T cells of MH and HO calves. Representative dot-plots depict IFN-γ+ T cell subsets in CD4+ (a), CD8+ (B) T cells and total lymphocytes (C). Representative histograms showed the expressions of IFN-γ in lymphocytes (D), CD4+ (E) and CD8+ (F) T cells in the whole blood of MH and HO calves. Data were shown in LSM ± SEM (n = 25). *p < 0.05, **p < 0.01.

Article Snippet: Monoclonal antibodies (mAb) specific for bovine TCRγδ (WC1), CD8, CD4 and IFN-γ with different fluorochrome conjugates were purchased from BIO-RAD (BIO-RAD, Shanghai, China) and mAb to human CD27, mouse CD44 were purchased from Biolegend (Biolegend, San Diego, USA) (Table 1).

Techniques: Expressing

Journal: The veterinary quarterly

Article Title: Holstein × Montbéliarde-sired F1 generation crossbred female calves have an increased cellular immune response potential compared with purebred Holsteins.

doi: 10.1080/01652176.2024.2435982

Figure Lengend Snippet: Figure 6. The expression of IFN-γ was derived from subsets of CD4+ and CD8− T cells, characterized by CD44+ cells rather than CD44− cells. Representative dot-plots depict CD4+ (A) and CD8- (B) T cells subsets of IFN-γ and CD44 from MH (left panel) and HO (right panel) calves. Numbers represent the percentages of cells in each quadrant. Bar graphs showed the expressions of IFN-γ by CD44+ or CD44− subsets in CD4+ (C) and CD8+ (D) T cells of MH and HO calves. Data were shown in LSM ± SEM (n = 25). Labels (a, b, c and d) of different letters indicated a significant difference between any two different sets of data (p < 0.01).

Article Snippet: Monoclonal antibodies (mAb) specific for bovine TCRγδ (WC1), CD8, CD4 and IFN-γ with different fluorochrome conjugates were purchased from BIO-RAD (BIO-RAD, Shanghai, China) and mAb to human CD27, mouse CD44 were purchased from Biolegend (Biolegend, San Diego, USA) (Table 1).

Techniques: Expressing, Derivative Assay

Journal: Annals of Translational Medicine

Article Title: Accumulation of TNFR2-expressing regulatory T cells in malignant pleural effusion of lung cancer patients is associated with poor prognosis

doi: 10.21037/atm-20-7181

Figure Lengend Snippet: List of the antibodies

Article Snippet: APC mouse anti-human IFN-γ , B27 , AB_398580 , 554702 , BD Sciences.

Techniques: Purification

Journal: Annals of Translational Medicine

Article Title: Accumulation of TNFR2-expressing regulatory T cells in malignant pleural effusion of lung cancer patients is associated with poor prognosis

doi: 10.21037/atm-20-7181

Figure Lengend Snippet: Effects of blocking TNFR2 on effector CD8+T cell function present in MPE. PEMCs from MPE were cultured in medium alone, anti-TNFR2 mAbs, or isotype IgG. On day 3, intracellular staining was performed following a 5-hour stimulation of PEMCs with PMA (50 ng/mL) and ionomycin (1 µg/mL). Four hours into stimulation, Brefeldin A was added at a final concentration of 3 µg/mL to block cytokine secretion. (A) Representative FACS analysis of IFN-γ+ cells in CD8+T cells after 72 h at the indicated conditions, and (B) summary data are shown (n=4). (C) Comparison of IFN-γ concentrations in the 72-h supernatants of PEMCs exposed to anti-TNFR2 mAbs or Isotype IgG (n=4). Flow analysis was gated on live CD3+CD8+ cells. Data are expressed as means ± SEM; **, P<0.01, ***, P<0.001 by paired Students t-test. TNFR2, tumor necrosis factor receptor type II; MPE, malignant pleural effusion; PEMC, pleural effusion mononuclear cell.

Article Snippet: APC mouse anti-human IFN-γ , B27 , AB_398580 , 554702 , BD Sciences.

Techniques: Blocking Assay, Cell Function Assay, Cell Culture, Staining, Concentration Assay, Comparison

Journal: Science Advances

Article Title: SARS-CoV-2 receptor binding domain displayed on HBsAg virus–like particles elicits protective immunity in macaques

doi: 10.1126/sciadv.abl6015

Figure Lengend Snippet: ( A ) Design of the nonhuman primate study in cynomolgus macaques. ( B ) Titers of RBD-specific antibody binding in animal sera. ( C ) Titers of SARS-CoV-2 pseudovirus–neutralizing antibody in animal sera. NT 50 , end point of 50% reduction of virus expression. ( D ) Expression of IFN-γ from cells stimulated with S1 protein peptides. Statistical significance was determined by a Kolmogorov-Smirnov test. n.s., not significant ( P > 0.1). Black bars represent median values. Gray dotted line represents limit of detection. PBMCs, peripheral blood mononuclear cells.

Article Snippet: Plates were coated with mouse anti-human IFN-γ antibody (BD Pharmigen) at 5 μg per well and incubated at 4°C overnight.

Techniques: Binding Assay, Expressing