Journal:

Article Title: Herpes Simplex Virus Type 1-Specific Cytotoxic T-Lymphocyte Arming Occurs within Lymph Nodes Draining the Site of Cutaneous Infection

doi:

Figure Lengend Snippet: Vβ10 expression by CD8+ CD25+ activated T cells from the draining lymph nodes of HSV-1-infected mice determined directly ex vivo or after 3 days of culture without exogenous antigen. Three mice were infected in the hind footpads with 4 × 105 PFU of HSV-1, and 5 days later the cells from the draining popliteal lymph nodes were collected, viable cell counts were performed, and 1 × 106 to 2 × 106 cells were placed into culture without exogenous antigen. Another 106 cells were triple stained with Vβ10, CD25, and CD8 antibodies prior to analysis by flow cytometry. After 3 days the cultured cells were similarly analyzed by flow cytometry. Dead cells were excluded by using propidium iodide staining. The dot plots represent CD8 versus CD25 staining for the popliteal lymph node cells ex vivo, and after 3 days in culture without exogenous antigen (in vitro). The histogram shows the Vβ10 receptor expression of the CD8+ CD25+ T cell subsets. The percentage of CD8+ CD25+ cells in the lymph nodes, and the percentage of Vβ10+ T cells among the CD8+ CD25+ T cell subsets are shown as a mean and standard deviation for the three mice analyzed.

Article Snippet: Lymph node cells cultured for 3 days without exogenous antigen were also stained with allophycocyanin-labeled anti-CD8 (53-6.7; Pharmingen) and FITC-labeled anti-CD25 (PC61.5.3; Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada).

Techniques: Expressing, Infection, Ex Vivo, Staining, Flow Cytometry, Cell Culture, In Vitro, Standard Deviation

Journal:

Article Title: Herpes Simplex Virus Type 1-Specific Cytotoxic T-Lymphocyte Arming Occurs within Lymph Nodes Draining the Site of Cutaneous Infection

doi:

Figure Lengend Snippet: The CD8+ CD25+ T cells from the draining lymph nodes of HSV-1-infected mice analyzed after 3 days of culture without exogenous antigen are predominantly gB specific. A mouse was infected in the hind footpads with 4 × 105 PFU of HSV-1, and 5 days later the cells from the draining popliteal lymph nodes were collected and placed into culture without exogenous antigen. After 3 days, the cultured cells and lymph node cells isolated from a naive mouse were triple stained with CD25 and CD8 antibodies, and the H-2Kb/gB tetramer and analyzed by flow cytometry. Dead cells were excluded by using propidium iodide staining. The dot plot represents CD8 versus CD25 staining for the cultured popliteal lymph node cells. The histograms show the level of H-2Kb/gB tetramer staining of the CD8+ CD25hi and CD8+ CD25lo T cell subsets from the cultured (shaded) and naive (unshaded) lymph node cell populations.

Article Snippet: Lymph node cells cultured for 3 days without exogenous antigen were also stained with allophycocyanin-labeled anti-CD8 (53-6.7; Pharmingen) and FITC-labeled anti-CD25 (PC61.5.3; Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada).

Techniques: Infection, Cell Culture, Isolation, Staining, Flow Cytometry

Journal: Molecular Medicine Reports

Article Title: Effects of combined treatment with PD-L1 Ig and CD40L mAb on immune tolerance in the CBA/J × DBA/2 mouse model

doi: 10.3892/mmr.2020.10977

Figure Lengend Snippet: Expansion of the CD4 + CD25 + T-cell population is driven by combined treatment with PD-L1 Ig and anti-CD40L mAbs in the CBA/J × DBA/2 model. (A) Number of CD4 + CD25 + T cells in splenocytes was determined by flow cytometry. (B) Gating strategy used for flow cytometry, using the FSC/SSC method. R1 represents the T lymphocyte population. (C) Percentage of CD4 + CD25 + T cells. ***P<0.001 vs. the normal group (CBA/J × BALB/c). ### P<0.001 vs. the spontaneous abortion group (CBA/J × DBA/2); &&& P<0.001 vs. PD-L1 Ig + CD40L mAb group. PD-L1, programmed death-ligand 1; Ig, immunoglobulin; CD40L, CD40 ligand; mAb, monoclonal antibody; FSC, forward scatter; SSC, side scatter; PE, phycoerythrin.

Article Snippet: The splenic cells were labeled with both anti-mouse CD25 FITC (cat. no. BG-07312-50-100; BioGems) and anti-mouse CD4 PE (cat. no. 85-12-0041-81; eBioscience; Thermo Fisher Scientific, Inc.) in the dark at 4°C for 30 min.

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

doi: 10.3390/ijms26073236

Figure Lengend Snippet: Lidocaine induced the secretion of IL-10, TGF-β, and IL-35 by CD4 + CD25 + and CD14 + PBMCs and inhibited the secretion of IL-10, TGF-β, and IL-35 by CD4 + CD25 + and CD14 + TIICs. The effect of lidocaine on the secretion of IL-10 ( A , B ), TGF-β ( C , D ), and IL-35 ( E , F ) from CD4 + CD25 + and CD14 + PBMCs and tumor-infiltrating immune cells (TIICs) was investigated. CD4 + CD25 + and CD14 + PBMCs or TIICs were cultured in the absence or presence of graded concentrations of lidocaine (from 0.25 mM to 1.5 mM). The levels of IL-10, TGF-β, and IL-35 in the supernatant were determined at 72 h by ELISA. Cell viability was >95%, as assessed by trypan blue exclusion. Data are presented as the mean ± SEM from three independent experiments, each performed in duplicate, using distinct samples (PBMCs) or three different donors (TIICs). * p < 0.05, ** p < 0.01. The values marked as 0 without lidocaine represent the basal levels of IL-10, TGF-β, and IL-35 in CD4 + CD25 + PBMCs and CD14 + PBMCs.

Article Snippet: Reagents and sources were as follows: lidocaine (Xylocaine ® 2% for Intravenous Injection) (Water for injections) (Cenexi, Fontenay-sous-Bois, France); FITC-conjugated IgG1 anti-human CD14 (BD Bioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD8 (eBioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD25 (Elabscience, San Diego, CA, USA); IgG1-FITC isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA); PE-conjugated IgG1 anti-human CD4 (Elabscience, San Diego, CA, USA); PE-conjugated IgG1 anti-human IFN-γ (Elabscience, San Diego, CA, USA); IgG1-PE isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

doi: 10.3390/ijms26073236

Figure Lengend Snippet: Lidocaine inhibited IL-35, IL-10, and TGF-β production by CD4 + CD25 + Foxp3 + and CD4 + CD25 + CD127 − TIICs. Sorted CD4 + CD25 + TIICs were treated with 1.5 mM lidocaine for 72 h followed by staining with anti-IL-35, anti-IL-10, anti-TGF-β, and anti-Foxp3 antibodies for flow cytometry analysis. Isotype controls were used to distinguish between positive and negative cells for IL-35, IL-10, TGF-β, and Foxp3. Typical flow cytometry dot plot analysis revealed the percentage of ( A , B ) CD35 + Foxp3 + CD4 + CD25 + TIICs, ( C , D ) IL-10 + Foxp3 + CD4 + CD25 + TIICs, and ( E , F ) TGF-β + Foxp3 + CD4 + CD25 + TIICs treated with lidocaine (1.5 mM). ( G , H ) The expression levels of CD127 in lidocaine-treated IL-35 + CD4 + CD25 + , IL-10 + CD4 + CD25 + , and TGF-β + CD4 + CD25 + cells were analyzed using flow cytometry. The expression levels are shown on histograms. Isotype controls were used to distinguish between positive and negative cells for CD127. Cell viability was >95%, as assessed by trypan blue. RMFI: relative mean fluorescence intensity. Data are from distinct samples and presented as the mean± SEM in three different experiments, each performed in duplicate. * p < 0.05, ** p < 0.01.

Article Snippet: Reagents and sources were as follows: lidocaine (Xylocaine ® 2% for Intravenous Injection) (Water for injections) (Cenexi, Fontenay-sous-Bois, France); FITC-conjugated IgG1 anti-human CD14 (BD Bioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD8 (eBioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD25 (Elabscience, San Diego, CA, USA); IgG1-FITC isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA); PE-conjugated IgG1 anti-human CD4 (Elabscience, San Diego, CA, USA); PE-conjugated IgG1 anti-human IFN-γ (Elabscience, San Diego, CA, USA); IgG1-PE isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA).

Techniques: Staining, Flow Cytometry, Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

doi: 10.3390/ijms26073236

Figure Lengend Snippet: Schematic diagram illustrating the lidocaine-mediated anti-tumoral mechanism through immunogenic cell death targeting PGCCs. Lidocaine inhibited the production of IL-35, IL-10, and TGF-β by CD4 + CD25 + Foxp3 + tumor-infiltrating immune cells (TIICs). Additionally, lidocaine enhanced anti-tumor immunity by reducing PD-1 expression and increasing IFN-γ expression on CD8 + TIICs via the NF-κB signaling pathway. The lidocaine-treated CD8 + TIICs subsequently promoted the immunogenic cell death of primary gastric cancer cells (PGCCs). Long→: treated. Short→: linked. ┬: inhibition.

Article Snippet: Reagents and sources were as follows: lidocaine (Xylocaine ® 2% for Intravenous Injection) (Water for injections) (Cenexi, Fontenay-sous-Bois, France); FITC-conjugated IgG1 anti-human CD14 (BD Bioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD8 (eBioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD25 (Elabscience, San Diego, CA, USA); IgG1-FITC isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA); PE-conjugated IgG1 anti-human CD4 (Elabscience, San Diego, CA, USA); PE-conjugated IgG1 anti-human IFN-γ (Elabscience, San Diego, CA, USA); IgG1-PE isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA).

Techniques: Expressing, Inhibition

Journal: STAR Protocols

Article Title: Protocol for the isolation of mouse senescence-associated CD4 + T cells using flow cytometry and functional assays

doi: 10.1016/j.xpro.2023.102472

Figure Lengend Snippet: Ab staining panel B to isolate “enriched” CD153 + SA-T cells

Article Snippet: Anti-CD25 (PC61.5), FITC (1:200) , Tonbo Biosciences , Cat# 35-0251; RRID: AB_2621685.

Techniques: Staining, Concentration Assay

Journal: PLoS ONE

Article Title: Forsythoside A Inhibits BVDV Replication via TRAF2-Dependent CD28–4-1BB Signaling in Bovine PBMCs

doi: 10.1371/journal.pone.0162791

Figure Lengend Snippet: PBMCs were collected at 12 h after stimulation. (A) Representative flow cytometry dot plots, (B) Flow cytometry was used to determine the percentage of CD3 + CD25 + population in the bovine PBMCs. Data are presented as means ± SEM of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following antibodies were used: Alexa Fluor 647-conjugated rat anti-human CD3 (Bio-Rad, Hercules, CA) and isotype control Alexa Fluor 647 conjugated-rat IgG1 (BD Biosciences) FITC-conjugated mouse anti bovine CD25 (GeneTex, San-Antonio, TX) and isotype control mouse FITC-conjugated mouse IgG1 (BD Biosciences) For each reaction, 2 × 10 6 cells were suspended in 50 μl of 0.1% bovine serum albumin (BSA)-PBS (wash buffer) and stained and cells were selected by gating based on size and granularity to exclude other cells and cellular debris.

Techniques: Flow Cytometry