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: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: FARSB promotes tumorigenesis and anti-tumor immunity in LUAD. A, qRT-PCR analysis of FARSB mRNA levels in different cell groups (si-NC, control group; si-FARSB, FARSB knockdown group). B, WB analysis of FARSB protein expression in various cell groups. C, CCK-8 assay evaluating the effect of FARSB knockdown on cell viability. D, EdU assay assessing the impact of FARSB knockdown on cell proliferation. E and F, Flow cytometry analysis of the effect of FARSB knockdown on cell apoptosis. G, WB analysis of PD-L1 (an immune checkpoint protein) expression levels. H, CFSE fluorescence staining assessing CD8 + T cell proliferation (CFSE dye dilution measures CD8 + T cell division). I-K, Flow cytometry analysis of IFN-γ (I), GZMB (J), and TNF-α (K) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8 + T cell cytotoxic function. N = 3, *** p < 0.001 and **** p < 0.0001 denote statistical significance. CFSE = carboxyfluorescein succinimidyl ester; FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; PD-L1 = programmed death-ligand 1; TNF-α = tumor necrosis factor-alpha; WB = western blot.

Article Snippet: Antibodies included: PE Anti-Human interferon-gamma (IFN-γ) Antibody (E-AB-F1196, Elabscience, Wuhan, China), Granzyme B (GZMB) Monoclonal Antibody (GB11, APC, Thermo Fisher), and tumor necrosis factor-alpha (TNF-α) Monoclonal Antibody (MAb11, APC, 17-7349-82, Thermo Fisher).

Techniques: Quantitative RT-PCR, Control, Knockdown, Expressing, CCK-8 Assay, EdU Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: FARSB Activates the mTOR signaling pathway, promoting LUAD tumorigenesis and anti-tumor immunity. A, GSEA analysis of enriched pathways associated with differential FARSB expression in LUAD. B, WB analysis of the expression of the pathway-related proteins p-mTOR, mTOR, p-p70S6K, p70S6K, p-4EBP1, and 4EBP1. C, CCK-8 assays evaluating cell viability. D, EdU assays assessing cell proliferation. E, Flow cytometry analysis of cell apoptosis. F, WB analysis of PD-L1 expression levels. G, CFSE fluorescence staining assessing CD8 + T cell proliferation. H-J, Flow cytometry analysis of IFN-γ (H), GZMB (I), and TNF-α (J) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8 + T cell cytotoxic function. n = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001 denote statistical significance. CFSE = carboxyfluorescein succinimidyl ester; FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; mTOR = mammalian target of rapamycin; ns = no significant differences; PD-L1 = programmed death-ligand 1; TNF-α = tumor necrosis factor alpha; WB = western blot.

Article Snippet: Antibodies included: PE Anti-Human interferon-gamma (IFN-γ) Antibody (E-AB-F1196, Elabscience, Wuhan, China), Granzyme B (GZMB) Monoclonal Antibody (GB11, APC, Thermo Fisher), and tumor necrosis factor-alpha (TNF-α) Monoclonal Antibody (MAb11, APC, 17-7349-82, Thermo Fisher).

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: SPI1 Downregulates FARSB to modulate the mTOR signaling pathway, thus influencing LUAD progression and anti-tumor immunity. A, qRT-PCR analysis of FARSB and SPI1 mRNA levels in different cell groups. B, WB analysis of FARSB and SPI1 protein expression. C and D, WB analysis of mTOR signaling pathway-related proteins. E, CCK-8 assays evaluating cell viability. F, EdU assays assessing cell proliferation. G, Flow cytometry analysis of cell apoptosis. H, WB analysis of PD-L1 expression levels. I: CFSE fluorescence staining assessing CD8 + T cell proliferation. J-L: Flow cytometry analysis of IFN-γ (J), GZMB (K), and TNF-α (L) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8+ T cell cytotoxic function. n = 3, *** p < 0.001, **** p < 0.0001 denote statistical significance. FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; mTOR = Mammalian target of rapamycin; ns = no significant differences or significant correlation.; PD-L1 = programmed death-ligand 1; qRT-PCR = quantitative reverse transcription-polymerase chain reaction; TNF-α = tumor necrosis factor alpha; WB = western blot.

Article Snippet: Antibodies included: PE Anti-Human interferon-gamma (IFN-γ) Antibody (E-AB-F1196, Elabscience, Wuhan, China), Granzyme B (GZMB) Monoclonal Antibody (GB11, APC, Thermo Fisher), and tumor necrosis factor-alpha (TNF-α) Monoclonal Antibody (MAb11, APC, 17-7349-82, Thermo Fisher).

Techniques: Quantitative RT-PCR, Expressing, CCK-8 Assay, Flow Cytometry, Fluorescence, Staining, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Immune escape between endoplasmic reticulum stress-related cancer cells and exhausted CD8+T cells leads to neoadjuvant chemotherapy resistance in ovarian cancer.

doi: 10.1016/j.bbrc.2024.150686

Figure Lengend Snippet: Fig. 8. Inhibition of NECTIN2 expression in ID8 cells can promote the cytotoxic function of co-cultured CTLs. a The expressions of PD1, PRF1 and IFNγ in CTLs cocultured with ID8 cells were detected by flow cytometry, n = 3. b-d Histogram plots show the proportion of PD1+ CTLs (b), PRF1+ CTLs (c) and IFNγ+ CTLs (d) in all CTLs co-cultured with ID8 cells in each group, n = 3.

Article Snippet: The transfected ID8 cells were co-cultured with the CTLs following stimulation by 8.5 μM cisplatin or the same volume of PBS (control group) for 24 h. CTLs in the supernatant were collected and the expression of depletion marker PD1(PE/DazzleTM 594 anti-mouse CD279 (PD1) Antibody, 109116, Biolegend, USA) and cytotoxic marker PRF1 (APC anti-mouse Perforin Antibody, 154304, Biolegend, USA) and IFNγ (FITC Anti-Mouse IFNγ Antibody, E-AB-F1101UC, elabscience, China) were detected by flow cytometry.

Techniques: Inhibition, Expressing, Cell Culture, 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