Journal: The Journal of International Medical Research

Article Title: Prognostic significance of CEA, Ki67 and p53 in pseudomyxoma peritonei of appendiceal origin

doi: 10.1177/03000605211022297

Figure Lengend Snippet: Univariate analysis of overall survival of patients ( n = 141) with pseudomyxoma peritonei.

Article Snippet: Immunohistochemical staining was performed using primary mouse monoclonal antibodies against Ki67 (clone UMAB107 ready to use; OriGene China, Beijing, China), p53 (clone DO7 ready to use; OriGene China) and CEA (clone COL1 ready to use; OriGene China) that were processed in automated staining equipment (INTELLIPATH FLX®; Biocare Medical, Pacheco, CA, USA).

Techniques:

Journal: The Journal of International Medical Research

Article Title: Prognostic significance of CEA, Ki67 and p53 in pseudomyxoma peritonei of appendiceal origin

doi: 10.1177/03000605211022297

Figure Lengend Snippet: The difference in clinicopathological characteristics between patients ( n = 141) with pseudomyxoma peritonei stratified according to Ki67 and p53 immunostaining levels.

Article Snippet: Immunohistochemical staining was performed using primary mouse monoclonal antibodies against Ki67 (clone UMAB107 ready to use; OriGene China, Beijing, China), p53 (clone DO7 ready to use; OriGene China) and CEA (clone COL1 ready to use; OriGene China) that were processed in automated staining equipment (INTELLIPATH FLX®; Biocare Medical, Pacheco, CA, USA).

Techniques: Immunostaining

Journal: Frontiers in Immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: Downregulation of IRF4 enhances CAR T cell functionality. (A) CAR T cells (starting with 1 × 10 5 CAR T cells) underwent four rounds (R1-R4) of stimulation with GFP-labeled CEA + BxPC-3 cells (1 × 10 5 tumor cells at the beginning of each round). At the end of each round, CAR T cells (live CD3 + CAR + ) (left panel) and BxPC-3 cells (right panel) were quantified by flow cytometry. Data represent means ± SEM of six donors, p values were calculated by Student´s t test, ns indicates not significant, and * indicates p ≤ 0.05. (B-G) Phenotypic analysis of CD8 + CAR T cells during repetitive antigen stimulation. CAR T cells underwent three rounds (R1-R3) of antigen-stimulation with unlabeled BxPC-3 cells. At the end of each round, the CD8/CD4 T cell ratio was determined (B) . CAR T cells were stained for CD8 and further characterized regarding TIM-3 (C) , PD-1 (D) , TIGIT (E) expression and effector-memory cell differentiation: SCM = T stem-cell-memory (CD45RO + CD62L + ), EM = effector-memory (CD45RO + CD62L - ), CM = central-memory (CD45RO + CD62L + ), E = effector (CD45RO - CD62L - ) (F) , and CD27 expression (G) . Data represent geometric means of ± SEM of at least four donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤ 0.05, **indicates p ≤ 0.01.

Article Snippet: The density of CEA on the surface of 293T cells, BxPC3-cells and MIA PaCa-2 cells was determined using the QuantiBRITE PE assay (LOT: #60550, BD) according to the manufacturer ́s instructions in conjunction with a PE-labeled anti-CEA antibody (clone TET2, Miltenyi) using flow cytometry.

Techniques: Labeling, Flow Cytometry, Staining, Expressing, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: Downregulation of IRF4 in CAR T cells enables killing of targets with low antigen density. (A) Staining of target cells MIA PaCa-2 for CEA expression using an APC Vio 770-conjugated anti-CEA antibody. One representative staining out of three experiments is shown. (B) T cells (1 × 10 5 T cells) with and without IRF4 downregulation were co-culture with GFP-labeled CEA + MIA PaCa-2 cells (1 × 10 5 tumor cells). After three days, CEA + MIA PaCa-2 cells (live GFP + cells) and CAR T cells (live CD3 + CAR + ) (C) were counted by flow cytometry using counting beads. Data represent means ± SEM of five donors, p values were calculated by Student’s t test, ns: not significant, **p ≤ 0.01. (D) Cytotoxicity of CAR T cells upon a 72-hour co-culture with MIA PaCa-2 cells or CEA - 293T cells was measured at the indicated effector to target cell ratios by an XTT-based colorimetric assay. Data represent means ± SEM of four donors, p values were calculated by Student´s t test. (E) IFN-γ and IL-2 in the supernatants after a 48-hour co-culture with medium (w/o), 293T cells, and MIA PaCa-2 cells was recorded by ELISA. Data represent means ± SEM of three donors, p values were calculated by Student´s t test, ns: not significant. (F) Staining of CAR T cells for CD25 and for pSTAT5 (G) after three days of co-culture with MIA-PaCa-2 cells. Data represent geometric means of ± SEM of three donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤ 0.05. (H) ELISA-based quantification of CAR-activation induced IFN-γ and IL-2 in the supernatant after a 48-hour culture on 96 well. plates coated with the anti-idiotypic monoclonal antibody BW2064/36 at the indicated concentrations. Data represent means ± SEM of eight donors, p values were calculated by paired t test, *p ≤ 0.05.

Article Snippet: The density of CEA on the surface of 293T cells, BxPC3-cells and MIA PaCa-2 cells was determined using the QuantiBRITE PE assay (LOT: #60550, BD) according to the manufacturer ́s instructions in conjunction with a PE-labeled anti-CEA antibody (clone TET2, Miltenyi) using flow cytometry.

Techniques: Staining, Expressing, Co-Culture Assay, Labeling, Flow Cytometry, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Activation Assay