Journal: iScience

Article Title: Membrane repair following filtroporation-induced cell permeabilization

doi: 10.1016/j.isci.2025.114317

Figure Lengend Snippet: Delivery and editing at the beta globin locus and RNA-Seq following the filtroporation-mediated permeabilization of human hematopoietic stem and progenitor cells (HSPCs) (A) Schematic of filtroporation-induced cell deformation and permeabilization. (B) Digital image of filtroporation system with 6 parallel reactions. Inset displays digital image of cell culture insert used for filtroporation. (C) Principal component analysis from RNA sequencing (RNA-Seq) studies. (D and E) Volcano plots of differentially expressed genes based on the RNA-Seq analysis of HSPCs 3 h post filtroporation (FP) or electroporation (EP). CD55-targeting Cas9 ribonucleoproteins (RNPs) were used as a model cargo. (F) Quantified pathway analysis matching keywords for each biological process category. (G) Percentage of insertions and deletions (INDELs) as determined by PCR, Sanger sequencing, and tracking of INDELs by the decomposition (TIDE) analysis of genomic DNA extracted from HSPCs pre-stimulated for 48 h at 4–5 days post-FP with RNPs designed to target the human beta globin (HBB) gene. Sources of HSPCs were either cord blood (CB) or peripheral blood mobilized HSPCs (PBSCs). Samples were either treated in the absence of cargo (Mock FP), FP-treated only with polyglutamic acid (PGA), FP-treated with RNPs formulated with increasing ratios of Cas9:sgRNA (1:1.2 to 1:2), or FP-treated with RNPs plus varying amounts of PGA (varied from 0.6:1 to 1:1 PGA:sgRNA volume ratios). Unless otherwise noted, the Cas9:sgRNA ratio was 1:1.2. (H) Viabilities of HSPCs corresponding to treatments in (G) at 24 h post-FP as determined by acridine orange/propidium iodide fluorescent staining. Data are shown for n ≥ 2 biologically independent donors from at least two independent experiments. (∗ p < 0.05) RNA-Seq data are obtained from triplicate samples in experiments with PBSCs from a single donor. (∗ p < 0.05). See also and . Data are shown as mean ± standard deviation.

Article Snippet: For PS bead experiments, fluorescent beads (Fluoro-Max Dyed Blue Aqueous Fluorescent Particles, Fisher Scientific, 09-980-484) were sonicated to break up aggregates and diluted to 30 μg/mL for filtroporation and nucleofection experiments (stock solution is 1% solids in buffer).

Techniques: RNA Sequencing, Cell Culture, Electroporation, Sequencing, Staining, Standard Deviation

Journal: iScience

Article Title: Membrane repair following filtroporation-induced cell permeabilization

doi: 10.1016/j.isci.2025.114317

Figure Lengend Snippet: Timed delivery of fluorescently tagged dextran to Jurkat cells (A) Schematic of typical and timed delivery experiments performed. In a typical experiment, target cells are premixed with fluorescent cargo (fluorescein isothiocyanate dextran, FITC-Dex). In timed delivery experiments, cells are treated in delivery buffer without cargo while FITC-Dex is added at a later time point. Flow cytometry is performed within 2 h of treatment to determine delivery efficiency (percentage of cells positive for FITC). (B) Filtroporation of Jurkat cells with FITC-Dex; cargo was either premixed or added at the specified time point. Controls were untreated, mock filtroporated (FP without cargo) or incubated with FITC-Dex without FP (Incub Ctrl). (C) Viabilities determined by 4′,6-diamidino-2-phenylindole (DAPI) staining at the time of flow cytometry after FP experiments. (D) Nucleofection (EP) of Jurkat cells with FITC-Dex; cargo was either premixed or added at the specified time point. Controls were untreated or mock nucleofected (EP without cargo). (E) Viabilities determined by 4′,6-diamidino-2-phenylindole (DAPI) staining at the time of flow cytometry after EP experiments. Data are shown for n ≥ 2 independent experiments. See also and . Data are shown as mean ± standard deviation.

Article Snippet: For PS bead experiments, fluorescent beads (Fluoro-Max Dyed Blue Aqueous Fluorescent Particles, Fisher Scientific, 09-980-484) were sonicated to break up aggregates and diluted to 30 μg/mL for filtroporation and nucleofection experiments (stock solution is 1% solids in buffer).

Techniques: Flow Cytometry, Incubation, Staining, Standard Deviation

Journal: iScience

Article Title: Membrane repair following filtroporation-induced cell permeabilization

doi: 10.1016/j.isci.2025.114317

Figure Lengend Snippet: Calcium concentration is critical during transfection by filtroporation (A) Delivery efficiency determined by flow cytometry within 2 h of filtroporation of Jurkat or CD34 + bone marrow derived human hematopoietic stem and progenitor cells (HSPCs) with fluorescein isothiocyanate (FITC)-tagged dextran (FITC-Dex) in cell media with low calcium (Roswell Park Memorial Institute, RPMI), FP-Dex (0.42 mM Ca 2+ ), or with additional 1 mM CaCl 2 added, FP-Dex (1.42 mM Ca 2+ ). Controls were either untreated, filtroporated without cargo (FP-Mock), or incubated in FITC-Dex without FP (Incub-Dex). (B) Viabilities of filtroporated Jurkat cells determined by 4′,6-diamidino-2-phenylindole (DAPI) at the time of flow cytometry. Cells were also transfected in phosphate buffered saline (PBS) as delivery buffer (FP-Dex-PBS). (C) Jurkat cells were filtroporated with green fluorescent protein (GFP)-encoding plasmid (pGFP) in different delivery buffers: RPMI, minimum essential media (MEM) containing no calcium, or a 1:1 mixture of RPMI and MEM (0.21 mM Ca 2+ ). Media was introduced at the bottom of the collection tube such that cells subjected to filtroporation fell into either RPMI, MEM or RPMI:MEM 1:1 media. Results are displayed for cells at 24 h post-filtroporation (timepoint with highest expression). (D) Cell counts at 24–72 h post-filtroporation performed with acridine orange/propidium iodide fluorescent staining. (E) Percentage of GFP-positive cells (left) and cell viability (right) determined by DAPI staining at the time of flow cytometry. Mock RPMI/MEM: cells filtroporated without cargo in RPMI or MEM; FP-RPMI-pGFP: cells subjected to FP in RPMI with pGFP cargo; FP-MEM-pGFP: cells subjected to FP in MEM with pGFP cargo; FP-MtR-pGFP: cells subjected to FP with pGFP cargo in MEM falling into RPMI media; FP-Premix-pGFP: cells subjected to FP with pGFP cargo in RPMI:MEM premixed media. Data are shown for n ≥ 2 independent experiments. (∗∗ p < 0.005, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001). Data are shown as mean ± standard deviation.

Article Snippet: For PS bead experiments, fluorescent beads (Fluoro-Max Dyed Blue Aqueous Fluorescent Particles, Fisher Scientific, 09-980-484) were sonicated to break up aggregates and diluted to 30 μg/mL for filtroporation and nucleofection experiments (stock solution is 1% solids in buffer).

Techniques: Concentration Assay, Transfection, Flow Cytometry, Derivative Assay, Incubation, Saline, Plasmid Preparation, Expressing, Staining, Standard Deviation

Journal: iScience

Article Title: Membrane repair following filtroporation-induced cell permeabilization

doi: 10.1016/j.isci.2025.114317

Figure Lengend Snippet: SNAP23 and CHMP4B are involved in membrane repair immediately after filtroporation (A–H) Confocal images overlaying enhanced green fluorescent protein (eGFP) and 4′,6-diamino-2-phenylindole (DAPI) fluorescence channels for untreated and filtroporated Jurkat cells expressing (A,E) GRAF1-eGFP, (B,F) SNAP23-eGFP, and (C,G) CHMP4B-mCherry, with (D,H) free cytoplasmic eGFP as a negative control. (I–P) Quantification of foci per cell per image captured on confocal microscopy for the different cell lines with cells immediately fixed after filtroporation or at different time points (GRAF1-eGFP: I and M; SNAP23-eGFP: J and N; CHMP4B-mCherry: K and O; free cytoplasmic eGFP: L and P). (∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). See also . Data are shown as mean ± standard deviation. Scale bars are 10 μm in panels A and E, and 5 μm in all other images.

Article Snippet: For PS bead experiments, fluorescent beads (Fluoro-Max Dyed Blue Aqueous Fluorescent Particles, Fisher Scientific, 09-980-484) were sonicated to break up aggregates and diluted to 30 μg/mL for filtroporation and nucleofection experiments (stock solution is 1% solids in buffer).

Techniques: Membrane, Fluorescence, Expressing, Negative Control, Confocal Microscopy, Standard Deviation