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: Human: Jurkat Clone E6-1 , ATCC , TIB-152.
Techniques: Concentration Assay, Transfection, Flow Cytometry, Derivative Assay, Incubation, Saline, Plasmid Preparation, Expressing, Staining, Standard Deviation