Affinipure f ab 2 fragment specific goat anti mouse igg antibody

Planar bilayers were formed by using small liposome droplets with clean glass coverslips as described previously. Briefly, biotin-labeled CD5 and CD7 proteins (ACRO system) were added to the bilayer and incubated for 30 min at 37 °C. After they were washed, 1.0 × 10⁵ CAR⁺ T cells of each group were suspended in T-cell medium and incubated with the lipid bilayers for 15 min at 37 °C. Then, the cells were fixed for 30 min with 4% paraformaldehyde at 4 °C. To visualize the immune synapse, F-actin was stained with Alexa Fluor 594-conjugated phalloidin (Abcam) following the manufacturer’s instructions. Subsequently, a FITC-conjugated AffiniPure F(ab)'2 fragment-specific goat anti-mouse IgG antibody (Jackson ImmunoResearch) or FITC-conjugated anti-gamma tubulin antibody (Abcam) was used to stain CARs or MTOCs, respectively. Nuclei were stained with Hoechst (Sigma) for 5 min. Confocal images were acquired by a Leica SP8 confocal laser-scanning microscope. All images were confirmed not to be overexposed by the software. All images were processed and analyzed with LAS X (Leica) and ImageJ (National Institutes of Health).

Peripheral blood samples were obtained from healthy volunteers approved by the Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (2023-0349). Informed consent from all participants was obtained. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation with Ficoll. T cells from PBMCs were activated with human T-cell activation beads (GenScript, L00899) in X-Vivo 15 medium at a density of 1 million/ml for 2 days and then expanded with 50 ng/ml IL-2 for several days, throughout which 2 μM GSK2879552 or vehicle was added.
The CD19-specific CAR construct containing FMC63 scFv, 4-1BB cytoplasmic domain, and CD3z cytoplasmic domain was obtained from Addgene (135992) and cloned into an EGFP-expressing lentiviral vector. Lentiviral particles were produced as described above and used to transduce pre-activated PBMCs by spin-infection. The expression of CD19-CAR was verified by flow cytometry after the sequential staining with a biotin-SP-AffiniPure F(ab)’2 fragment-specific goat anti-mouse IgG antibody (Jackson ImmunoResearch, 115-066-072) and streptavidin-phycoerythrin (BioLegend, 405203). Transduced cells were cultured and expanded in X-Vivo 15 medium supplemented with 50 ng/mL IL-2 for 6–8 days before in vitro assays or transfer into tumor-bearing NCG mice.

Gene‐edited Jurkat CAR19 and CAR T‐19 cells were incubated with a biotin‐SP‐AffiniPure F(ab)’2 fragment‐specific goat anti‐mouse IgG antibody (Jackson ImmunoResearch) to assess CAR expression, followed by incubation with streptavidin‐PE (BD Biosciences). In addition, the gene‐edited Jurkat CAR19 and CAR T‐19 cells were incubated with anti‐human B2M, CD3 (BD Biosciences), HLA‐ABC, HLA‐C, HLA‐E, and HLA‐G (Biolegend) antibodies.
Gene‐edited CAR T‐19 cells were washed and stained with anti‐human CD3, CD4, CD8, CD45RO, and CD62L antibodies (BD Biosciences).
Gene‐edited CAR T‐19 cells were cocultured with Raji or K562 cells at an effector‐to‐target (E:T) ratio of 1:1 together with an anti‐human CD107a antibody (BD Biosciences) for 1 h to assess degranulation, followed by incubation with a Golgi Plug protein transport inhibitor (BD Biosciences) for 3 h.
Primary NK cells were incubated with anti‐human CD3, CD56 (BD Biosciences), NKG2A, KIR2DL4, and LILRB1 (Biolegend) antibodies.
All of the antibody information is presented in Supporting Information Table 1. All samples were assessed using DxFLEX flow cytometry (Beckman Coulter). The data were analyzed with Kaluza Analysis 2.0 (Beckman Coulter) and FlowJo software version 10 (FlowJo LLC).