Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Gating strategy for the single-step method. Absolute counting beads were excluded based on scatter and fluorescence properties. Dead cells were excluded using 7-aminoactinomycin D (7-AAD). CD45-positive leukocytes were selected, and lymphocytes were identified according to side scatter (SSC) characteristics. CD3-positive T cells were gated, and CAR-T cells were defined as viable CD45+/CD3+/CAR+ events using directly fluorochrome-conjugated CAR detection reagents (CD19 or BCMA). CD4 and CD8 subpopulations were subsequently identified within the CAR-positive T-cell compartment. Absolute quantification was calculated using TruCount beads according to the manufacturer’s formula.

Article Snippet: New CAR detection reagent (CDR) directly coupled to fluorochrome, either BCMA (BCMA CDR-PE, Miltenyi Biotec 130-133-888) or CD19 (CD19 CDR α-FMC63-PE, Miltenyi Biotec 130-127-342) was added extemporaneously in the analytical tube.

Techniques: Fluorescence, Quantitative Proteomics

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Determination of LOD and LLOQ for the single-step method. CAR-T cell absolute counts were measured in 10 negative control samples from patients not treated with CD19 or BCMA CAR-T cells. The blue line represents the mean background signal. LOD was defined as mean + 3 SD and LLOQ (green dashed line) as mean + 10 SD.

Article Snippet: New CAR detection reagent (CDR) directly coupled to fluorochrome, either BCMA (BCMA CDR-PE, Miltenyi Biotec 130-133-888) or CD19 (CD19 CDR α-FMC63-PE, Miltenyi Biotec 130-127-342) was added extemporaneously in the analytical tube.

Techniques: Negative Control

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Timeline of T cell knock-in electroporation workflow, knock-in strategy and designs for BCMA-CAR (1.6 kb integration) across the series of DNA HDR template formats tested. (b) Comparison of BCMA-CAR HDRTs at concentrations 5nM-160nM or 0-100E3 MOI in terms of knock-in efficiency, (c) live cell count per 1e6 edited cells, and (d) knock-in cell count per 1e6 edited cells measured 7 days post electroporation. (e) Knock-in of a logic-gated synNotch circuit (5.6 kb integration) using linear ssDNA + tCTS, circular cssDNA + CTS, and nanoplasmid + CTS templates with corresponding knock-in efficiency, live cell count and knock-in cell count 7 days post electroporation using Cas9 mRNA. Circular cssDNA was produced and provided by Kano Therapeutics. (f) Knock-in strategy and designs for a logic-gated synNotch circuit (5.6 kb integration) at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation using Cas9 RNP or Cas9 mRNA. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. CTS, Cas9 target site. RNP, ribonucleoprotein. MOI, multiplicity of infection.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Knock-In, Electroporation, Comparison, Cell Characterization, Produced, Infection

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Knock-in strategy and designs for BCMA-CAR (1.6 kb integration) at concentrations 5nM-160nM with corresponding (b) knock-in efficiency, (c) live cell count, and (d) knock-in cell count 7 days post electroporation for the following templates respectively: dsDNA, dsDNA + tCTS, ssDNA, ssDNA + tCTS, circular ssDNA + CTS, circular ssDNA + mutated v1 CTS v1, circular ssDNA + mutated v2 CTS. Circular cssDNA was produced and provided by Touchlight. (e) Knock-in strategy and designs for CAR-CARD-11 (3.5 kb integration) using linear ssDNA + tCTS template or a nanoplasmid + CTS template at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Knock-In, Cell Characterization, Electroporation, Produced

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Designs for BCMA-CAR (1.6 kb integration) HDRTs variants tested and corresponding template copies per genome measured at 4 hours and 3 days post electroporation (b) and half-life measured by qPCR. (c) Template copies per genome measured over time at 4 hours, 2 days, 3 days, 5 days, 7 days, and 12 days with R2 and half-life values corresponding to each donor. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. tCTS, truncated Cas9 target site. CTS, Cas9 target site. NP, nanoplasmid. cssDNA, circular cssDNA.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Electroporation

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Timeline of T cell knock-in electroporation workflow, knock-in strategy and designs for BCMA-CAR (1.6 kb integration) across the series of DNA HDR template formats tested. (b) Comparison of BCMA-CAR HDRTs at concentrations 5nM-160nM or 0-100E3 MOI in terms of knock-in efficiency, (c) live cell count per 1e6 edited cells, and (d) knock-in cell count per 1e6 edited cells measured 7 days post electroporation. (e) Knock-in of a logic-gated synNotch circuit (5.6 kb integration) using linear ssDNA + tCTS, circular cssDNA + CTS, and nanoplasmid + CTS templates with corresponding knock-in efficiency, live cell count and knock-in cell count 7 days post electroporation using Cas9 mRNA. Circular cssDNA was produced and provided by Kano Therapeutics. (f) Knock-in strategy and designs for a logic-gated synNotch circuit (5.6 kb integration) at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation using Cas9 RNP or Cas9 mRNA. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. CTS, Cas9 target site. RNP, ribonucleoprotein. MOI, multiplicity of infection.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Knock-In, Electroporation, Comparison, Cell Characterization, Produced, Infection

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Knock-in strategy and designs for BCMA-CAR (1.6 kb integration) at concentrations 5nM-160nM with corresponding (b) knock-in efficiency, (c) live cell count, and (d) knock-in cell count 7 days post electroporation for the following templates respectively: dsDNA, dsDNA + tCTS, ssDNA, ssDNA + tCTS, circular ssDNA + CTS, circular ssDNA + mutated v1 CTS v1, circular ssDNA + mutated v2 CTS. Circular cssDNA was produced and provided by Touchlight. (e) Knock-in strategy and designs for CAR-CARD-11 (3.5 kb integration) using linear ssDNA + tCTS template or a nanoplasmid + CTS template at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Knock-In, Cell Characterization, Electroporation, Produced

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Designs for BCMA-CAR (1.6 kb integration) HDRTs variants tested and corresponding template copies per genome measured at 4 hours and 3 days post electroporation (b) and half-life measured by qPCR. (c) Template copies per genome measured over time at 4 hours, 2 days, 3 days, 5 days, 7 days, and 12 days with R2 and half-life values corresponding to each donor. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. tCTS, truncated Cas9 target site. CTS, Cas9 target site. NP, nanoplasmid. cssDNA, circular cssDNA.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Electroporation