Kaluza analysis 2

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).

A flow cytometry assay was performed as previously described [18 (link)]. After 48 h of transfection, the transfected cells in each well were collected and subjected to apoptosis detection using Annexin V-FITC/PI staining. Each sample was tested in triplicate, and analyses were performed using Kaluza Analysis 2.0 (Beckman Coulter, Inc.) according to the manufacturer’s guidelines.

K-562 cells were washed with PBS and labeled with 5 μM pacific blue succinimidyl ester (PBSE; Thermo Fisher). Per well, 1 × 10⁵ K-562 cells were co-cultured with GTA002 cells at E:T 1:1 in a total volume of 200 µL in a 96-well V-bottom plate. At the start of co-incubation, anti-CD107a PE (H4A3, BioLegend) was added at 1 μg/ml. After 5 h of incubation at 37°C, the cells were transferred to a V-bottom plate and stained with 4 μg/ml 7-AAD (Sigma) and 1 μg/ml anti-CD56 APC-A750 (N901, Beckman Coulter) in 25 μL. Read-out of the cytotoxicity assay was done using Cytoflex LX (Beckman Coulter), and data analysis was done using Kaluza Analysis 2.1 (Beckman Coulter). Killing was determined by the following formula: %cytolysis = 100-[(number of (7-AAD⁻/PBSE⁺) K-562 cells in co-culture/number of (7-AAD⁻/PBSE⁺) K-562 cells alone)*100]. Degranulation was calculated as % Δ degranulation = (% CD107a⁺/CD56⁺/7-AAD⁻ cells in co-culture) – (% CD107a⁺/CD56⁺/7-AAD⁻ cells in NK cells alone).

Harvested at the density of 10 × 10⁶, H9C2 cells were stained with H2DCFDA (MedChemExpress, China) to detect ROS level, and stained with Bodipy (BODIPY™ 581/591 C11, Invitrogen, USA) or Liperfluo (Dojindo, Shanghai, China) to detect lipid peroxidation level. The signals were collected using by flow cytometry (CytoFLEX S equipped with Kaluza analysis 2.1, Beckman coulter, USA) or imaged by an Olympus FV3000 confocal laser scanning microscope (Olympus, Japan).