Journal: bioRxiv

Article Title: Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

doi: 10.1101/2024.04.11.588248

Figure Lengend Snippet: Effect of hypoxia-preconditioned MSCs (MSCs HYP , MSCs HYP→APO , MSCs HYP+APO ) on the proliferation of aGVHD patients derived T-cell. The bar graph represents (A) the percentage of CD3 + CFSE + proliferating T-cell (n=5). (B) the ratio of CD4 + /CD8 + T cell (n=5) in the direct co-culture of MSCs and T-cell. Data shown as Mean±S.D.; Statistical analysis: Tukey’s multiple comparisons test; **≤0.01; ****≤0.0001. Abbreviations: BM: Bone marrow; WJ: Wharton’s Jelly; MSCs: Mesenchymal Stem Cells; HYP: Hypoxia-preconditioned; APO: Apoptosis .

Article Snippet: CD3 + T-cell was labeled with 1µM Cell Trace TM CFSE dye (BD Biosciences, USA) for 20 minutes at 37C and followed by activation of CD3 + T-cell with PHA (1µg/ml) (Sigma, USA) and IL-2 (50IU/ml) (Thermo Fisher Scientific, USA) in 1X Rosewell Park’s Memorial Institute (RPMI) medium supplemented with L-glutamine (Thermo Fisher Scientific, USA), 10% FBS, 1% antibiotic-antimycotic solution at 37C, 5% CO 2 for 48 hours.

Techniques: Derivative Assay, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: CD8 + T Cells Directed Against a Peptide Epitope Derived From Peptidoglycan-Associated Lipoprotein of Legionella pneumophila Confer Disease Protection

doi: 10.3389/fimmu.2020.604413

Figure Lengend Snippet: IFN-γ and CTL responses to PAL peptides. (A) To determine which peptides were able to induce IFN-γ production from PAL-specific spleen cells, mice were immunized with pcDNA3-PAL at 0, 1, and 2 weeks. At 3 weeks, the mice were sacrificed and the spleens were harvested. Six × 10 6 splenocytes were stimulated for 2 days at 37 o C with each of 13 peptides (P1–P13) at a final concentration of 5 µg/ml. The cell culture supernatants were collected and IFN-γ concentrations measured. n.d. (not detectable). * p < 0.05 compared to control, ** p < 0.05 compared to P6, *** p < 0.05 compared to P9. (B) We repeated the above experiments, except that the splenocytes were stimulated with P8 or P10 (as a control) at final concentrations of 1, 200, 1,000, or 10,000 ng/ml. * p < 0.05 compared to 1 ng/ml, ** p < 0.05 compared to 100 ng/ml, *** p < 0.05 compared to 1,000 ng/ml. (C) Mice were immunized and the spleens were obtained as above. In vitro lytic activity was measured using the splenocytes as effector cells and syngeneic targets (primed with P8 or rPAL) in the LDH release cytotoxicity assay, as described in the Methods and Materials . * p < 0.05 compared to control. (D) A CFSE-based cytotoxicity assay was performed to measure in vivo lytic activity, as described in the Methods and Materials . Cells with low and high density CFSE staining were gated and the CFSE intensity, as assessed by flow cytometry, was plotted. One representative result (% lysis) is shown. The values and bars represent mean IFN-γ concentrations and percent lysis and the SDs, respectively. * p < 0.05 compared to naïve mouse, ** p < 0.05 compared to CpG-ODN or P8.

Article Snippet: They were prepared by being divided into two tubes containing 2 × 10 7 cells/ml in RPMI-1640 with 2.5% FBS, and the fluorescent carboxylfluorescein diacetate succinimidyl ester (CFSE) dye (BD Bioscience) added at 2.5 µM (CFSE low ) or 20 µM (CFSE high ), then the cells were resuspended and incubated at 37°C for 40 min.

Techniques: Concentration Assay, Cell Culture, In Vitro, Activity Assay, Cytotoxicity Assay, In Vivo, Staining, Flow Cytometry, Lysis