elisa kits  (Novus Biologicals)

Journal: Clinical and Experimental Medicine

Article Title: IL-21- and CXCL9-engineered GPC3-specific CAR-T cells combined with PD-1 blockade enhance cytotoxic activities against hepatocellular carcinoma

doi: 10.1007/s10238-024-01473-2

Figure Lengend Snippet: Cytotoxicity assays in vitro. a GPC3 expression of HCC cell lines were analyzed by FCM. Isotype Ab served as a control. b GPC3 expression confirmed by Western blotting analysis. c The secretion of IL-21 or CXCL9 of indicated CAR-T cells determined by ELISA. d Cytokines (IL-2, IFN-γ and TNF-a) released by different CAR-T cells after 24 h coculture with Hep3B cells at 1:1 ratio was measured by ELISA. e Cytotoxicity of indicated CAR-T cells incubated with HepG2, Hep3B, Huh-7 and SK-Hep-1 at 5:1, 10:1, and 20:1 ratio for 24 h. Each data set reflected mean ± SD of triplicates, ns represent p > 0.05, ** p < 0.01 *** p < 0.001. The ELISA results were compared with one-way ANOVA, and the cytotoxicity assay was conducted by two-way ANOVA

Article Snippet: The ELISA kits for IL-2 and IFN-γ were purchased from Sino Biological (Beijing, China), and the ELISA kits for IL-21 and CXCL9 were purchased from Novus Biologicals (Littleton.

Techniques: In Vitro, Expressing, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Cytotoxicity Assay

Journal: Molecular Cancer Therapeutics

Article Title: SENTI-101, a Preparation of Mesenchymal Stromal Cells Engineered to Express IL12 and IL21, Induces Localized and Durable Antitumor Immunity in Preclinical Models of Peritoneal Solid Tumors

doi: 10.1158/1535-7163.mct-21-0030

Figure Lengend Snippet: Figure 2. Disclosure of SENTI-101. Lead candidate optimization, functional validation and construct map. A and B, Human BM-MSCs from two independent donors were engineered with constructs in which different promoters were driving EGFP expression. EGFP fluorescence was quantified using flow cytometry. % transduction (A) and mean fluorescence intensity (MFI; B) were compared across multiple cell lines and through multiple passages (up to day 28 post-transduction). C, Human BM-MSCs were engineered to overexpress the lead candidate of cytokines IL12 and IL21. SFFV or EF1a were the promoters driving protein expression. Cytokine production (pg/1e6 cells) was quantified by ELISA in supernatants collected 24 hours after seeding the cells. Results are shown as average of technical replicates (N ¼ 3). D, Different constructs were designed to enhance cytokine production by changing the signal sequence of IL21 (na€ve compared with no signal sequence, IL2 signal sequence or IL8 signal sequence). Cytokine production of IL12 or IL21 (pg/1e6 cells) was quantified by ELISA in supernatants collected 24 hours after seeding the cells. Results are shown as average and SEM of independent replicates (N ¼ 3). E, IL12 function was confirmed using a reporter cell line (IL12-HEK-Blue). Reporter cells were incubated with supernatants collected from different MSC engineered with the listed constructs. Recombinant cytokines were used as control. Results are shown as average of 4 technical replicates. F, IL21 function was confirmed using phospho-flow for the downstream targets pSTAT1 and pSTAT3. NK92 cells were incubated with supernatant from different MSCs engineered with the listed constructs and relative expression of pSTAT1 or pSTAT3 was quantified. Recombinant cytokines were used as control. Results are shown as average of technical replicates. G, Plasmid map of SB00880, our selected lead candidate construct for SENTI- 101. The construct encodes for IL12 and IL21 human sequences codon-optimized linked by a furin-T2A sequence and under the SFFV promoter and contains a Woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE) to enhance expression.

Article Snippet: Mouse and human ELISA kits for IL12 and IL21 were purchased from R&D Systems and were used following manufacturer’s instructions.

Techniques: Functional Assay, Biomarker Discovery, Construct, Expressing, Cytometry, Transduction, Enzyme-linked Immunosorbent Assay, Sequencing, Incubation, Recombinant, Control, Plasmid Preparation, Virus