easysep human cd3 positive selection kit ii  (STEMCELL Technologies Inc)

Journal: Science Advances

Article Title: Armored human CAR T reg cells with PD1 promoter-driven IL-10 have enhanced suppressive function

doi: 10.1126/sciadv.adx7845

Figure Lengend Snippet: ( A ) Schematic diagram of alloantigen-based suppression assay shown in (B) and (C). HLA-A2 + DCs were cocultured with HLA-A3 − T reg cells for 72 hours. Allogeneic CPD eFluor 450–labeled HLA-A3 + CD3 + responder T cells were then added, and cocultures were maintained for an additional 96 hours. ( B ) Representative CD4 + responder T cell proliferation, gated on live HLA-A3 + CPD eFluor 670 − CPD eFluor 450 + CD4 + cells. ( C ) % Suppression of CD4 + responder T cell proliferation, relative to responder T cells cultured without T reg cells. ( D ) Schematic diagram of islet antigen–based suppression assay shown in (F) to (H). HLA-A2 + HLA-DR4 + DCs were cocultured with T reg cells for 48 hours. CD4 + 4.13-TCR + responder T cells and 1 nM GAD65 peptide were then added, and cocultures were maintained for an additional 48 hours. ( E ) CD4 + 4.13-TCR + T cell proliferation following 96-hour coculture with immature HLA-DR4 + DCs and varying GAD65 peptide concentrations (without T reg cells). Control cells were pulsed with an irrelevant hemagglutinin peptide (100 nM). Division indices provided. ( F ) Representative CD4 + responder T cell proliferation, gated on live CD4 + CPD eFluor 670 − mTCRβ + cells. ( G ) % Suppression of responder T cell proliferation, relative to responder T cells stimulated without T reg cells. ( H ) Relative cytokine analysis from 1:128 T reg cell:responder T cell (T resp cell) ratio. TNFα, IL-17A & IL-17F were not detected. Averaged data are means ± SEM with non-linear regression lines ( n = 8 to 10). Statistical significance was determined using mixed-effects analysis with P values shown. n.s., not significant.

Article Snippet: After 3 days, CD3 + responder T cells (previously enriched from an HLA-A3 + individual using the EasySep Human CD3 Positive Selection Kit II; STEMCELL Technologies) were labeled with CPD eFluor 450 and added to the cocultures at a 5:1 responder T cell:DC ratio.

Techniques: Suppression Assay, Labeling, Cell Culture, Control

Journal: Experimental Hematology & Oncology

Article Title: Engineered circular RNA-based DLL3-targeted CAR-T therapy for small cell lung cancer

doi: 10.1186/s40164-025-00625-8

Figure Lengend Snippet: Optimization of electroporation conditions for circRNA expression in Human CD3 + T cells. A Comparison of EGFP expression in T cells electroporated with circRNA at different voltages (1400V, 1600V, and 1800V). B Flow cytometry analysis of anti-DLL3 CAR expression at varying electroporation voltages. C Evaluation of CAR expression and apoptosis rates in T cells electroporated with various concentrations of circRNA (1–6 µg/million cells). D Assessment of immunogenicity in circRNA and mRNA-transfected T cells under optimized electroporation conditions. E Assessment of CAR expression duration in circRNA and mRNA-transfected T cells. F Evaluation of cell proliferation in circRNA and mRNA-transfected T cells using the CCK-8 assay. G Evaluation of cell proliferation in circRNA and mRNA-transfected T cells using the CFSE assay. circRNA circular RNA, mRNA messenger RNA, EGFP enhanced green fluorescent protein, CAR-T chimeric antigen receptor T cell, DLL3 Delta-like Ligand 3, CCK-8 Cell Counting Kit-8, CFSE Carboxyfluorescein Succinimidyl Ester

Article Snippet: CD3 + T cells were then purified employing the EasySep TM Human CD3 Positive Selection Kit II (Stemcell, 17851).

Techniques: Electroporation, Expressing, Comparison, Flow Cytometry, Immunopeptidomics, Transfection, CCK-8 Assay, CFSE Assay, Cell Counting