anti cd69 pe vio770 conjugated  (Miltenyi Biotec)

Journal: Biomolecules

Article Title: Exploring the Biological Activity of a Humanized Anti-CD99 ScFv and Antibody for Targeting T Cell Malignancies

doi: 10.3390/biom14111422

Figure Lengend Snippet: Inhibitory effects of HuMT99/3 compared with mAb MT99/3 on T cell activation. Upon anti-CD3 mAb OKT3 activation, PBMCs were treated with or without 20 µg/mL of mAb MT99/3, HuMT99/3, or isotype-matched control Abs (mouse IgG or Human IgG). ( A ) CFSE dilution technique was used for cell proliferation analysis after activation for 5 days. ( B ) T cell activation-associated markers CD69 and CD25 expressions in CD3 + T cells were determined by direct immunofluorescence staining at 18 and 72 h, respectively. ( C ) Cytokine production in CD3 + T cells was determined by intracellular staining. All experiments were analyzed by flow cytometry. Each data point in the presence of the indicated antibody was normalized relative to its medium control as 100%. The bar graphs or scatter plots exhibit mean ± SD. Unpaired t -test was used for comparison; * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Treated cells were harvested after cultivation for 18 h then stained with FITC-conjugated anti-CD3 mAb (BD Biosciences) and PE-conjugated anti-CD69 mAb (immunotools, Friesoythe, Germany) or after cultivation for 72 h then stained with FITC-conjugated anti-CD3 mAb (BD Biosciences) and PE-conjugated anti-CD25 mAb (BioLegend).

Techniques: Activation Assay, Control, Immunofluorescence, Staining, Flow Cytometry, Comparison

Journal: Oncogene

Article Title: GPA33 expression in colorectal cancer can be induced by WNT inhibition and targeted by cellular therapy

doi: 10.1038/s41388-024-03200-3

Figure Lengend Snippet: A Schema of the pSLCAR-GPA33 and pSLCAR-CD19 plasmid designs. scFv single-chain variable fragment, V H variable heavy chain, V L variable light chain, TM transmembrane region, ICD intracellular domain. B GFP expression was analyzed by flow cytometry to evaluate the CAR plasmid transduction rate in Jurkat cells or PBMCs. Transduction rate in untreated controls without co-culture is shown. n = 4 for Jurkat cells; for PBMCs, n = 4 different donors were analyzed at n = 3 replications, mean ± SEM. Percentage of CD69-positive Jurkat T cells after co-cultivation with target cells for 24 h analyzed by flow cytometry. C Co-culture with GPA33-negative HEK293T and SW480, or GPA33-positive SW1222 cells; D Co-culture with SW1222 cells at varying effector:target ratios. n = 3, mean ± SEM; 1-way ANOVA with Bonferroni multiple comparison test. E Untransduced, CD19-CAR or GPA33-CAR transduced PBMCs were co-cultured at a 1:1 ratio with SW1222 cells for 48 h. The IL-2 and IFNγ concentrations in the supernatant were determined using ELISA. PBMCs from 4 different donors were analyzed at n = 3, mean ± SEM; 1-way ANOVA with Bonferroni multiple comparison test. F Untransduced or GPA33-CAR transduced PBMCs were co-cultured with HEK293T cells with or without pcDNA3.1-mGPA33 or pcDNA3.1-GPA33 transfection at a 1:1 ratio for 48 h. The IL-2 and IFNγ concentrations in the supernatant were determined using ELISA. PBMCs from n = 4 different donors were analyzed at n = 3 replications, mean ± SEM; 1-way ANOVA with Bonferroni multiple comparison test. G Representative live cell images of CD19-CAR or GPA33-CAR transduced PBMCs during 1:1 co‑cultivation with SW1222 target cells over 66 h. CAR T cells are green and marked with white arrows; SW1222 tumor cells were stained with red nuclear dye. Scale bar: 50 µm. H – J Co-culture of CD19-CAR or GPA33-CAR transduced PBMCs with indicated target cells over 66 h. Target cells are indicated by red nuclear fluorescence. HEK293T cells were transduced with pcDNA3.1-GPA33 to drive GPA33 expression (HEK293T-GPA33). Shown are the normalized green object counst for relative CAR T cell numbers ( H ), the normalized red object counts for relative target cell numbers ( I ) and overall confluency determined through brightfield imaging. *** P < 0.001.

Article Snippet: To measure CD69 levels on CAR T cells, 10.000 transduced Jurkat cells were co-cultivated with SW1222, SW480 or HEK293T target cells at 1:1, 1:0.1 or 1:0.01 effector:target ratio for 24 h. Jurkat activation was assessed by co-expression of GFP and CD69 using an anti-human PE-conjugated CD69 antibody (Invitrogen), as previously described [ ].

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Transduction, Co-Culture Assay, Comparison, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Staining, Fluorescence, Imaging